Recommended Posts

I continue to be fascinated with the possible practical applications of all of this research, particularly with respect to CR practice. Like Dean, I am particularly interested in optimizing glucose control / glucose homeostasis. The study Dean highlighted above, where they found that high BAT individuals had an almost immediate glucose lowering or stabilizing BAT activation upon feeding makes me want to try the following experiment, this time with a control:

However there is FAR more going on with BAT than simply glucose control. It has become apparent through the research that a host of cell signaling pathways associated with longevity are involved.

Regarding the points being made about mice and room/lab temps. I got the sense from the back and forth as well as from reading some of the related publications, that there is quite a bit of uncertainty and/or opposing views on the matter even among researchers. My thoughts are that perhaps the focus is misplaced - if you want to compare mice to humans, we should be looking at and comparing the temps at which mice and humans achieve some level of BAT activation as the benchmark for comparison - this would provide a reproducible and more definitive answer to the debate in a way that is directly relevant to the science most of us are interested in (longevity). Also every scenario could be accounted for (bedding materials or lack thereof, clothing or lack thereof, singly or group housed, BMI, genetics, etc).

Regards,

Gordo

Edited March 16, 2016 by Gordo

Share this post

Link to post

Share on other sites

I really need to thank you for letting me know that your data from November 2002 was also used in the reference (2) paper Dean, since I was also in the group tested at that time. And for your results for your glucose versus testosterone in Fig 3 of (2) were in contracts to mine with glucose AUC of 120, testosterone of 16.5 and IGF01 of 76.5 (I have not T3 value) were exceptional and greatly different from my values. I was 97 pounds at the time.

Share this post

Link to post

Share on other sites

I'm still working on my "magnum opus" (or should I call it my 'Albatross' ☺) in response to Michael's skepticism. Hopefully I'll get it finished tomorrow...

In the meantime here is a nice popular press story about the Lee et al. paper (doi:10.1016/j.cmet.2016.02.007) that Michael posted and that I discussed a couple posts earlier in this thread. The article focuses on the potential to use brown fat to prevent diabetes.

Here are a few highlights and quotes from the lead author (Lee):

"We think brown fat may be functioning as a glucose buffer, smoothing out the fluctuations of glucose," endocrinologist Dr Paul Lee of Sydney's Garvan Institute of Medical Research said.

Interestingly, the researchers found brown fat activity rose at dawn just as people awoke.

"We speculate this may have an evolutionary origin because our ancestors had to go out and hunt in the mornings, which tended to be cold," Dr Lee said. "This brown fat and temperature boost may have helped prepare them."

Previous research has shown that the amount of brown fat we have increases with prolonged exposure to mildly cold temperatures (19 degrees Celsius) and decreases with prolonged exposure to warmer temperatures.

Dr Lee said that meant modern humans could actually be reducing their brown fat.

"Perhaps by not exposing ourselves to cold because of widespread central heating and clothing, this may also be contributing to diabetes."

That last one hits the nail on the head, as far as I'm concerned!

Its funny, the author uses the same old excuse used to motivate CR mimetic research, but in this case in the context of CE (Cold Exposure) rather than CR:

Dr Lee said it was not practical to boost brown fat by exposing ourselves to the cold, but that he and his colleagues were now trying to find out how the body itself switches on brown fat.

"Whichever signals that switch on this brown fat biorhythm may become a drug target in the future," he said.

It seems CE and CR are in the same boat in at least this one respect - both require too much discipline to expect the general population to engage in them!

--Dean

Share this post

Link to post

Share on other sites

Cold Exposure Albatross (Albatross-CE) - To make it easy to search for this post in the future...

Michael,

First off, I want to thank you for engaging in this discussion. I really appreciate your challenges to my interpretation of the scientific data surrounding cold exposure (CE), which is really getting quite extensive. It is, paradoxically, a really hot area, it would seem. It's only through such challenges that we can identify and get past our implicit assumptions and biases, and hopefully get closer to the truth.

Before getting into a detailed response to your skepticism, let me see if I can summarize my hypothesis about cold exposure & BAT, so you have something more specific to express reservations about, and to focus my response in the remainder of this post.

Cold Exposure Hypothesis:

Cold exposure in mammals (including humans) promotes health and longevity by several mechanisms, including, but not limited to, simply burning extra calories to create a net calorie deficit that helps the organism avoid obesity and hence avoid the negative health consequences that accompany it. If combined with eating limited calories and/or exercise, CE can make significant contributions to producing a net calorie deficit that is sufficiently large to cause the body to kick into a CR-like 'hunkered down' metabolic state of heightened repair and maintenance. At least some of these benefits are likely to result from an increased amount of brown adipose tissue (BAT) or more generally, an increased expression of uncoupling proteins (UCPs) in organs, fats and muscle tissues that cause mitochondria to burn calories in order to generate heat rather than make ATP for useful energy, and in the process potentially make the mitochondria operate more 'cleanly' - i.e. without generating as many ROSs (free radicals). But like the multiple evolutionary adaptations that kick in as a result of the metabolic stress of CR, CE is likely to trigger many different hormonal / physiological / biochemical responses that together allow the organism to survive the metabolic stress of CE.

There is a stronger speculation that I'm not ready to fully endorse because there isn't enough evidence either way. Namely that at least some of the benefits observed for CR will only manifest if the animal also exposed to cold housing conditions, and perhaps only if those CR animals have BAT or some other mechanism for active thermogenesis. Put the other way around, this speculation is that housing mammals at a comfortable, thermoneutral temperature so as not to require thermogenesis will blunt or erase at least some of CR's benefits.

With that on the table, let me now try to summarize your perspective, and especially your reservations / skepticism about this hypothesis, and see if I can provide sufficient evidence to overcome your skepticism.

First, something we appear to agree on - the obesity-prevention effects of increased energy expenditure as a result of CE will in all likelihood have health benefits, independent of any more direct benefits from CE-induced metabolic/hormonal/biochemical adaptations. Given what I understand to be the SENS perspective on aging, namely that aging simple is the accumulation of damage which largely results from the so-called "diseases of aging" (several of which in turn are brought on by obesity), it continues to confuse me how in your estimation delaying the onset and progression of the diseases of aging remains just a benefit to health, and isn't also conveying longevity benefits (i.e. slowing the aging process). For example, the 7th Day Adventists eat well & exercise, and thereby avoid obesity, CVD, diabetes, sarcopenia, cognitive decline, by slowing down accumulation of the damage resulting from a subset of what SENS characterizes as the Seven Causes of Aging, which manifest themselves symptomatically as the diseases of aging just listed. So as a result of this slowed accumulation of damage, the 7DAs live longer. Seems straightforward to me. But that is a topic for another thread where I hope you will indeed respond soon as promised, but in which one of your recent posts suggests you don't see quite as sharp a division between health-promoting and longevity-promoting interventions as I'd previously thought you did.

You also seem to acknowledge that colder ambient temperatures will result in cooler body temperatures, which will in turn likely slow down the onset and progression of cancer, something that rodents are particularly prone to and that CR in rodents does a good job of preventing, as long as housing temperatures are below thermoneutrality (more on that below).

Now on to where we seem to disagree, at least in part.

You wrote:

It seems clear, therefore, that the mice housed at 30°C in PMID 9032756 are really rather toasty, and those at room temperature are only very modestly cooler-housed than a human in normal room temperatures.

We could quibble over what the appropriate housing temperatures for mice should be, as Speakman & Keijer do with their critics in this series of back and forth and back again letters-to-the-editor about the their paper (PMID 24024125). Even Speakman acknowledges that cool-housed mice (20-22 °C) are thermally stressed:

At no point in our paper do we state that mice are ‘most comfortable’ at 20–22 °C, and the contention of our article was not, and was never stated to be, that ‘there is no distressing impact of cold temperatures on mice’

Your use of the colloquial "rather toasty" is ambiguous. By toasty do you mean "comfortably warm" or "heat stressed"? From a metabolic perspective, the evidence strongly favors the former interpretation (toasty = comfortably warm). Check out these two figures from [1], a huge review on rodent metabolic response to temperature variations:

As is apparent, across various strains of mice, their metabolic rate is minimized at around 30°C. Similarly, as can be seen from this graph also from [1], that the amount of BAT mass mice possess is minimized at 30 °C, and climbs steadily as temperature is reduced.

The author of [1] addresses the weird apparent uptick in BAT above 30 °C with the observation that it doesn't actually appear to be BAT that is increasing at the high temperatures:

The weight of BAT increased in a near linear fashion as temperature decreased below 30 °C. Interestingly, BAT weight also increased in mice exposed to extremely warm temperatures of 35 and 37.5 °C, environments that are well above the limits of normothermy for mice and must have certainly been stressful. It was noted that histologically, the BAT in warm adapted mice differed markedly from cold acclimated mice. BAT of cold exposed mice was highly developed and multilocular. BAT of the warm acclimated mice was unilocular, looking more like white adipose tissue.

To put this mice data in perspective, here is a helpful graphic from [4] illustrating the change in metabolic rate of mice, naked humans and lightly clothed humans as a function of temperature:

According to the above graphic, a mouse has a metabolic rate approximately 250% of it's baseline at a "normal" indoor temperature (20-22 °C). With a little extrapolation, you can see that the same boost in metabolic rate for a human would require exposure to 10°C (50°F), and that's if they are naked. Clothed it would be much lower...

To summarize, from a metabolic perspective, it appears that thermal stress for mice is minimized at around 30 °C, and is increased dramatically when mice are housed at normal lab temperatures (20-22 °C), as reflected in both a higher metabolic rate and higher BAT content. As a result, at "normal" indoor temperatures (20-22 °C), mice are quite thermally stressed, while humans are not, which isn't surprising, since we are the ones who get to define what "normal" is. ☺

So, getting back to your post, the bulk of the evidence seems to contradict you statement:

It seems clear, therefore, that the mice housed at 30°C in PMID 9032756 are really rather toasty, and those at room temperature are only very modestly cooler-housed than a human in normal room temperatures. This merits CR folk bearing a certain amount of temperature discomfort, but doesn't imply that the benefits are abolished if we aren't either exposed to the autumn elements naked or wearing ice vests.

I'm glad to see we're on the same page that some amount of temperature discomfort is likely to be beneficial for CR folks. That seems like an important takeaway message that we both agree on. It seems like now we're just haggling over price.☺

We saw above, mice living at 20-22 °C are metabolically quite challenged relative to living at 30 °C. But what about subjectively? The interesting question you pose is what amount of thermal discomfort might be the human equivalent of what mice experience at "normal" lab temperatures?

Obviously you can't ask mice directly "how cold are you?" but fortunately there have been behavioral studies that address this very issue - thermal preferences in mice. Specifically, if given their druthers (i.e. if they can move freely between cages of different temperatures), mice of several strains preferred a cage with a temperature of 26-29°Ceven with bedding material and two cage mates to huddle with [2]. So the typical 20-22°C housing temperature used in PMID 9032756 and virtually all CR experiments is a full 6.5°C (12°F) cooler than the temperature that mice prefer. On top of that, mice in CR experiments (including PMID 9032756) are housed singly - so no huddling or cuddling :-( as the mice in [2] were able to do to keep warm, and which (i.e. huddling) was shown in [1] to result in a nearly 50% reduction in BAT activity when mice were housed with just one other mouse rather than singly.

Michael, I'm not sure what temperature you find most comfortable when dressed in light clothing, but if you are like most people, it's probably in the neighborhood of 21°C (70°F). Subtract 6.5°C (12°F) from that and you get about 14.5°C (58°F). Try hanging out at 14.5°C (58°F) in light clothing for a few hours (to say nothing of 24/7 for your entire life) before suggesting that the mice aren't very thermally stressed at "normal" lab temperatures.

So the evidence is really quite solid - rodents housed at normal lab temperatures are quite thermally stressed, both metabolically and subjectively. The authors of [11] express it amusingly in the title of their paper - "Why We Should Put Clothes on Mice" and [12] even includes a helpful graphic illustrating how to improve the human-relevance of rodent lab experiments ☺:

But I have a different solution. Rather than warming up mice (with clothing or otherwise) so they have crappy health & longevity like we warm humans, why not cool ourselves down so we have good health & longevity like the thermally-challenged CR mice in PMID 9032756, who ate 20% more food, weighed the same, but lived longer (both median and max lifespan) than warm-housed CR mice?

Then you wrote:

... the renowned Dr. John Speakman, author of numerous provocative studies on CR and related subjects and the lead author of (1), will be one of our many exciting scientific presenters.

That is great news, especially since Dr. Speakman is the lead author of another, even more interesting and relevant paper I'll discuss below... [inserted later - actually to be discussed in an upcoming post...]

Next you quibble over whether mitochondrial biogenesis (i.e. formation of new mitochondria) is actually occurring in the BAT of CRed mice in PMID 18593277, as the authors suggest. This too seems like a red herring. Does it really matter whether the mechanism for increased BAT thermogenic capacity in CRed mice results from an increase in the number of mitochondria, the size of mitochondria or the enrichment of existing mitochondria with UCP1? Regardless of the mechanism, what the study showed was that CRed mice had ~60% less BAT than controls, but the mitochondria of the BAT they did retain was highly enriched (by about a factor of 2x) with heat-producing UCP1 protein. Hence the authors reference to "conservation of BAT thermogenic capacity" in CRed mice despite dramatic reduction in their total BAT tissue mass.

Just to translate this into layman's terms - in order to keep their skinny little butts warm in their cold, lonely cages (I can picture Sthira is seething now...), the CR mice in PMID 18593277 were able to cold-adapt by boosting the ability of the what little BAT they retained to generate even more heat than usual.

You then complain (ok, observe☺) that CR doesn't increase metabolic rate (i.e. metabolic activity per unit metabolically-active mass), and so an increase in burning of calories through BAT-driven thermogenesis appears ruled out. If you look at the table of tissue masses in my post about PMID 18593277, you'll see that while the change in BAT mass between controls and CR mice is dramatic in relative terms (i.e. CR mice have 60% less BAT than control mice), the fraction of the total body weight that BAT represents is tiny - 0.1% in CR mice vs. 0.15% in control mice. A very little amount of BAT can go a long way, especially when your BAT is supercharged like the CRed mice in this study.

So consider the following and tell me if it doesn't fit the data (it may not, I'm just asking):

CR mice have little body fat and a higher surface-to-volume ratio than control mice, meaning if anything they'll have to work (i.e. burn calories) at least as hard (and probably harder) to stay warm in their cool cages.

Fortunately per PMID 18593277, they have a small amount of supercharged BAT to do just that.

But since, as you observe, their overall metabolic rate remains relatively constant, they must be reducing metabolic expenditure elsewhere - either via reductions in other metabolic processes or in physical activity, in order to burn calories via thermogenesis in BAT or muscle cells.

In other words, mightn't the heretofore paradoxical lack of decrease in body-mass-normalized metabolic rate in CR mice (despite reduced body temperature) be explained by a shift in energy expenditure from "dirty" (ROS-producing) metabolic activities in other tissues to "clean-burning" uncoupled mitochondrial respiration in their tiny amount of supercharged BAT or in their muscles, in a desperate and only partially successful attempt by the CR mice to keep warm?

Put another way, mightn't part of the metabolic program that kicks in with CR in cold housing conditions be a relative increase in calorie expenditure devoted to clean-burning thermogenesis in order to keep warm? Seems pretty plausible to me.

Then you wrote:

I just can't mechanistically see how increasing BAT quantity or activity would exert an effect on aging per se.

This suggests to me a lack of imagination, or a reluctance to think very hard about it.☺

Here are a few prefatory remarks before diving into the details that constitute the bulk of this post.

First, as I mentioned above and in my last post, I'm never quite sure what you mean by "aging per se" (see this thread for discussion). But I'm going to assume you mean "accumulation of damage resulting from metabolic processes".

Next, as my Cold Exposure Hypothesis at the top makes explicit, it is the body's metabolic response to CE in general that I postulate to convey health and longevity benefits. Increased BAT and BAT activity is just one of many metabolic responses that could be involved (more on those below). It's like CR in this regard; many evolutionarily conserved pathways may be involved in the benefits of CE. This brings to mind another pro-survival stress response involving cold exposure - the mammalian dive reflex (MDR), the evolutionarily conserved ability of many mammals (including humans) to conserve oxygen as a result of cold water contacting the face and thereby increase survival. If we've preserved the MDR adaptation to cold water exposure, it seems pretty plausible to me that we'd preserve a highly-tuned, multifaceted metabolic response to an even more common, temperature-related and potentially life-threatening environmental stress, cold exposure. Parallels between the MDR and the body's response to more modest cold exposure will have to wait for another post...

What I'll do now is attempt to counter your skepticism about the anti-aging potential of CE & BAT by enumerating some of the mechanisms by which the research suggests they may indeed have health and longevity-promoting effects.

Obesity Avoidance - In your previous post you seem to poo-poo CE's (and specifically BAT's) ability to help prevent obesity/overweight as not a legitimate mechanism for slowing aging. This seems strange to me, and again gets back to your squirrelly definition of aging. It seems pretty clear that white adipose tissue, particularly abdominal WAT, is a very active endocrine organ, spewing out pro-inflammatory proteins that result in quite a bit of downstream aging-related damage. No? On top of that, the evidence in rodents (discussed here, here and here), dogs (discussed here), monkeys (discussed here, here and here), and humans (e.g. these discussions of Adventists and postmenopausal women), suggests that mostof CR's benefits result from quite mild calorie restriction - i.e. avoiding obesity/overweight on a healthy diet. So if that's what the evidence says is happening with CR, than CE & BAT could very well have the same positive effect on aging. In short, BAT's ability to help people avoid obesity, and so avoid the negative health & aging consequences that accompany obesity, could by itself serve as a legitimate mechanism by which having extra BAT impacts aging.

Improved Glucose Metabolism - I won't dwell on this one, since I addressed it in depth here yesterday in my response to your posting of Lee et al (doi:10.1016/j.cmet.2016.02.007). Suffice it to say that it appears that in humans, increased BAT levels is associated with improved glucose control, and effect that probably accounts for the reduced glycated hemoglobin (HbA1c) that occurs population-wide in winter months.

Improved Gut Microbiome - I won't dwell on this one either, since I addressed it in depth here and here. The basic upshot is that based on the results of PMID 26638070, chronic cold exposure alters the bacterial strains in the gut microbiome so as to make the digestive system more efficient at extracting calories, as well as improve insulin sensitivity and turn white fat into browning/beige fat, at least in rats. Several of us cold-exposed human CR practitioners show a similar gut bacteria profile to these cold-adapted rats, based on our uBiome.com results, as discussed here and here.

Reduced Core Temperature - One mechanism by which CR is thought to extend lifespan is through reduction in body temperature ([15] is a good review of this evidence). Study [9] found that reducing core body temperature of ad lib fed mice via genetic mutation that modulates hypothalamus temperature (the body's thermostat) and thereby reduces core body temperature, increases lifespan without changes in calorie intake. Depending on its severity, CE can easily reduces core body temperature even more than CR, and so is likely to extend lifespan along this same pathway.

Reduced Cancer Proliferation - Relatedly, I think we're in agreement on this one, although I agree it may not be a direct result of BAT per se. Consistent CE reduces body temperature (even with BAT working to keep warm), which appears associated with a reduction in the rate of cancer proliferation, as observed in [3] (full text). As you suggest, this is probably the way that cool-housed CR mice lived longer than warm-housed CR mice in PMID 9032756, and possibly [9] as well. But see the post referenced in the next item (immunocompetence) for another pathway actively upregulated by CE that dramatically reduces (by 60%) the rate of cancer grown.

Improved Immunocompetence - I won't dwell on this one here, since in this post (written after the one you're reading now), I go into great detail on research showing that CE (alone or in combination with exercise) appears to improves the immune system's ability to fight off invaders, including cancer, by elevating norepinephrine and interleukin-6, which in turn boost natural killer (NK) immune cells.

Reduced Inflammation - In another striking strike against thermoneutrality, this very recent study [7], found that housing mice at thermoneutrality (30°C) resulted in a dramatic increase in systemic inflammation and atherosclerosis relative to cool-housed (22°C) mice:

Mice housed at thermoneutrality develop metabolic inflammation in adipose tissue and in the

In other words, simply living in thermally-neutral, comfortable-for-a-mouse conditions resulted in increased inflammation which promoted atherosclerosis independent of obesity and insulin sensitivity.

Reduced Atherosclerosis - Speaking of CE's impact on atherosclerosis, Al and I went back and forth on this topic for a while earlier in this thread. I feel pretty confident I won that one ☺. Here is the post where I summarize the debate and point to this 2016 review [22] which summarizes the relationship between BAT and atherosclerosis as follows:

Improved Bone Health - Bone loss and the resulting increase in fracture risk is a serious concern as one ages. One troubling side effect of CR is a reduction in bone mineral density. Although there is some hope CR bones are lighter but not more fracture-prone, bone health remains a serious concern for CR practitioners. Fortunately increased levels of BAT are associated with increased bone mineral density, independent body weight. In other words, you can be thin with good bone density, as long as you've got BAT, as I discuss in detail here.

Reduced Oxidative Damage - Cold exposure reduces coupling in mitochondrial respiration, not just in BAT (via UCP1), but also in other tissues/organs, including muscles, via several different uncoupling proteins. Modest reductions in mitochondrial coupling is known to reduce the proton gradient substantially, which in turns results in reduced production of damaging ROSs (free radicals). So it seems to me a pretty clear story for aging-related benefits of CE accruing via this mechanism, although again it isn't specifically a result of BAT, but CE more generally. Suggestively, I talk in this post about two studies that found several genetic mutations that result in increased expression of several UCPs (including UCP1) are associated with extreme longevity in humans. In that same post I talk about how one of the few genetic mutations that set the long-lived naked mole rat apart from other rodents, and other mammals, is a mutation that upregulates expression of UCP1. Coincidence? Maybe, but maybe not, especially in light of a couple other studies by Speakman, that I was going to include here but that are important enough to merit their own post, especially considering how long this post is getting already. Stay tuned...

FGF-21 Production - I'll repeat and expand a bit on what I said in my post to which you are responding about the significance of FGF-21 as a pathway towards increased longevity. Cold exposure upregulates circulating fibroblast growth factor 21 (FGF21) in people [23] by 37%, and FGF21 in mice BAT by a factor of 40x [5]. Here is a a very nice graph from [6] that shows that in mice, not just BAT but plasma levels of FGF21 is unaffected by only 6h of cold exposure, but doubles after 24h, and triples after 30 days of CE:

So what good is FGF21 you ask? PMID 23066506 found that transgenic mice that overexpress FGF21 lived 40% longer than controls without the mutation, despite the FGF21-mutant mice eating a bit more than the normal mice. It's an argument in several steps (cold exposure → ↑ BAT → ↑ FGF21 → ↑ longevity), but it shows obesity-independent benefits of cold exposure & BAT. See this post for more discussion.

Interestingly, in addition to (or by way of) extending lifespan, it is well known that FGF21 improves insulin sensitivity & glucose metabolism [5]. And as we saw above, [6] shows in rats that cold exposure increased plasma FGF21, which helped with glucose control. We saw from Lee et al that in BAThigh humans, changes in glucose clearance occur subsequent to increased BAT activity, rather than proceeding it, and the authors were at a loss to say why/how. This results suggests that what could be going on in BAThigh humans is that FGF21 release by active BAT into the bloodstream improves glucose clearance a few minutes later. An intriguing possibility.

Adiponectin Production - Like CR [10], cold exposure increases adiponectin levels. Two hours of cold exposure resulted in a 70% increase in circulating adiponectin in adult men [36]. Study [8] found centenarians and their offspring had genetic mutations that boost adiponectin, and had higher circulating adiponectin, suggesting to the authors "their [i.e. adiponectin-promoting gene mutations] may promote increased lifespan through the regulation of adiponectin production and/or secretion." Study [35] found the same thing in a group of centenarian women - "As compared to BMI-matched [young, ~28 year-old] female controls, female centenarians had significantly higher plasma adiponectin concentrations. In addition, high concentrations of plasma adiponectin in centenarians was associated with favorable metabolic indicators, and with lower levels of C-reactive protein and E-selectin". For those of us who aren't lucky enough to have adiponectin-boosting genes, we can increase adiponectin levels via CR, cold exposure, or both. This video illustrates how wearing the Cool Fat Burner for two hours raises a cold-adapted person's adiponectin level by a whopping 62%!

Irisin Production - As discussed here and here, both cold exposure and exercise increase circulating irisin [23]. Irisin improves insulin sensitivity, increases bone quality and quantity [24], is involved in the building of lean muscle mass, and helps reduce obesity by converting white fat to brown fat. In short, it appears CE provides many of the same benefits of exercise, including raising irisin, without the sweating ☺. As a bonus, [25] found that:

healthy centenarians are characterized by increased serum irisin levels, whereas levels of this molecule were found to

be significantly lower in young patients with myocardial infarction. Our findings may prompt further research into the role

played by irisin not only in vascular disorders but also in life span modulation.

SIRT1 Pathway Activation - SIRT1 is upregulated by CR, and is thought to be one of the important pathways through which CR extends lifespan [27]. While the lifespan effects of SIRT1 in mammals are somewhat controversial, transgenic mice that overexpress SIRT1 have increased lifespan [30] and genetic mutations that result in elevated SIRT1 levels in people are associated with increased human longevity [31]. SIRT1 (and the other sirtuins) have many metabolic effects, but an important one for improving health and longevity is the fact that SIRT1 increases insulin sensitivity and glucose control in skeletal muscles [28], triggers the browning of white fat [32] and increases BAT activity [29], which is fully reversed by thermoneutral housing [29]. Mice partially lacking SIRT1 due to a genetic mutation experience BAT degeneration, reduced thermogenesis, increased inflammation and develop obesity and insulin resistance as a result [34]. Guess what - it's not just CR that upregulates SIRT1. Cold exposure increases SIRT1 phosphorylation/activity in both skeletal muscle and BAT, increasing thermogenesis and insulin sensitivity [33]. In short, both CR and CE upregulate SIRT1, which (may) increase lifespan, at least in part by improving insulin sensitivity in muscles and increasing BAT & BAT activity.

Improved Heat Tolerance / Increased Heat Shock Protein Expression - While it's not clear heat shock proteins (HSPs) increase lifespan in mammals like they do in lower organisms (like C elegans), HSPs are definitely important for stress resistance [18], and in particularly for the ability to cope with heat stress, hence the name. CR preserves HSP induction in response to thermal stress in aging mice [20], and prevent the age-related decline in several heat shock protein [21], and especially HSP70 [19] and HSP90 [21]. So might the cold of CE reduce one's heat shock protein levels, and ruin one's ability to cope with high temperatures? Nope. Quite the opposite in fact.

Study [13] found that the induction of three important heat shock proteins, Hsp70, Hsp90 and Hsp110 was higher in tissues of mice housed at 22 °C than mice housed at thermoneutrality (30°C) following 6 h of heat stress (i.e. high temperatures) which elevated core body temperature to 39.5 °C. So it looks like relative to living at comfortable, thermoneutral temperatures, CE helps with thermal tolerance at both extremes - hot and cold, by improving heat shock protein induction. Interestingly, [14] found increased expression of one of Hsp70 is associated with improved insulin sensitivity in monkeys and humans - "higher levels of [...] HSP70 protect against insulin resistance development during healthy aging." Review article [16] is a good discussion of heat shock proteins and lifespan in general, and [17] is a study that shows genetic mutations in heat shock proteins may be associated with improved human survival.

FInally, since this section is about the association of CE and BAT with longevity, I'd be remiss if I didn't mention the fact that three of the longest-lived small mammals, grey squirrels (24 yrs), bats (30 yrs), and naked mole rats (32 yrs), all have remarkably high levels of BAT and BAT activity, which is suggestive, but obviously not conclusive, evidence that BAT promotes health & longevity.

To wrap up, this diagram from [15]:

is missing a few important pathways discussed above, and suggests too strongly that reduced body temperature is the sole mechanism by which CE operates. Nevertheless, it illustrates how CR and CE activate many of the same health and longevity-promoting pathways. In fact it shows how CR piggybacks on many of the same temperature / thermogenic pathways that are more directly activated via CE. It also clearly illustrates how exposure to warm conditions could interfere with many CR benefits, by raising body temperature and counteracting CR's beneficial modulation of many different pathways.

I think the evidence summarized in this diagram, and the more comprehensive evidence presented in the entirety of this post (and the rest of the 129 posts in this thread!), illustrate that cold exposure is a legitimate alternative/adjunct to CR for potentially improving health and extending human lifespan.

In terms of total number of publications, the laboratory mouse (Mus musculus) has emerged as the most popular test subject in biomedical research. Mice are used as models to study obesity, diabetes, CNS diseases and variety of other pathologies. Mice are classified as homeotherms and regulate their core temperature over a relatively wide range of ambient temperatures. However, researchers find that the thermoregulatory system of mice is easily affected by drugs, chemicals, and a variety of pathological conditions, effects that can be exacerbated by changes in ambient temperature. To this end, a thorough review of the thermal physiology of mice, including their sensitivity and regulatory limits to changes in ambient temperature is the primary focus of this review. Specifically, the zone of thermoneutrality for metabolic rate and how it corresponds to that for growth, reproduction, development, thermal comfort, and many other variables is covered. A key point of the review is to show that behavioral thermoregulation of mice is geared to minimize energy expenditure. Their zone of thermal comfort is essentially wedged between the thresholds to increase heat production and heat loss; however, this zone is above the recommended guidelines for animal vivariums. Future work is needed to better understand the behavioral and autonomic thermoregulatory responses of this most popular test species.

----------------

[2] PLoS ONE 7(3): (2012) e32799. doi:10.1371/journal.pone.0032799

Heat or Insulation: Behavioral Titration of Mouse Preference for Warmth or Access to a Nest.

In laboratories, mice are housed at 20–24uC, which is below their lower critical temperature (<30uC). This increased thermal stress has the potential to alter scientific outcomes. Nesting material should allow for improved behavioral thermoregulation and thus alleviate this thermal stress. Nesting behavior should change with temperature and material, and the choice between nesting or thermotaxis (movement in response to temperature) should also depend on the balance of these factors, such that mice titrate nesting material against temperature. Naı¨ve CD-1, BALB/c, and C57BL/6 mice (36 male and 36 female/strain in groups of 3) were housed in a set of 2 connected cages, each maintained at a different temperature using a water bath. One cage in each set was 20uC (Nesting cage; NC) while the other was one of 6 temperatures (Temperature cage; TC: 20, 23, 26, 29, 32, or 35uC). The NC contained one of 6 nesting provisions (0, 2, 4, 6, 8, or 10g), changed daily. Food intake and nest scores were measured in both cages. As the difference in temperature between paired cages increased, feed consumption in NC increased. Nesting provision altered differences in nest scores between the 2 paired temperatures. Nest scores in NC increased with increasing provision. In addition, temperature pairings altered the difference in nest scores with the smallest difference between locations at 26uC and 29uC. Mice transferred material from NC to TC but the likelihood of transfer decreased with increasing provision. Overall, mice of different strains and sexes prefer temperatures between 26–29°C and the shift from thermotaxis to nest building is seen between 6 and 10 g of material. Our results suggest that under normal laboratory temperatures, mice should be provided with no less than 6 grams of nesting material, but up to 10 grams may be needed to alleviate thermal distress under typical temperatures.

----------

[3] Mech Ageing Dev. 1996 Nov 29;92(1):67-82.

A tumor preventive effect of dietary restriction is antagonized by a high housing

FGF21 is a novel metabolic regulator involved in the control of glucosehomeostasis, insulin sensitivity, and ketogenesis. The liver has been consideredthe main site of production and release of FGF21 into the blood. Here, we showthat, after thermogenic activation, brown adipose tissue becomes a source ofsystemic FGF21. This is due to a powerful cAMP-mediated pathway of regulation ofFGF21 gene transcription. Norepinephrine, acting via β-adrenergic, cAMP-mediated,mechanisms and subsequent activation of protein kinase A and p38 MAPK, inducesFGF21 gene transcription and also FGF21 release in brown adipocytes. ATF2 bindingto the FGF21 gene promoter mediates cAMP-dependent induction of FGF21 genetranscription. FGF21 release by brown fat in vivo was assessed directly byanalyzing arteriovenous differences in FGF21 concentration across interscapularbrown fat, in combination with blood flow to brown adipose tissue and assessmentof FGF21 half-life. This analysis demonstrates that exposure of rats to coldinduced a marked release of FGF21 by brown fat in vivo, in association with areduction in systemic FGF21 half-life. The present findings lead to therecognition of a novel pathway of regulation the FGF21 gene and an endocrine roleof brown fat, as a source of FGF21 that may be especially relevant in conditionsof activation of thermogenic activity.

Background: Evidence from experimental models of longevity indicates that maintenance of energy homeostasis could be indispensable for longevity across various species. In humans, it has been reported that maintenance of glucose homeostasis and vascular stability is one biomedical feature of centenarians, who have reached the maximum life-span. We hypothesized that adiponectin, a novel anti-inflammatory adipocytokine, could be a protective factor against age-related metabolic alteration and atherogeneity in centenarians.

Results: As compared to BMI-matched female controls, female centenarians had significantly higher plasma adiponectin concentrations. In addition, high concentrations of plasma adiponectin in centenarians was associated with favorable metabolic indicators, and with lower levels of C-reactive protein and E-selectin. In contrast, genetic analysis of 10 single nucleotide polymorphism (SNP) at adiponectin locus did not show significant association between the adiponectin gene variation and longevity.

Conclusions: Our results suggested that hyperadiponectinemia in centenarians could play a role in maintenance of energy homeostasis and vascular stability, and may contribute to longevity.

Link to post

Share on other sites

After that huge post, while waiting for Michael's response, I figured I'd share a short and relatively straightforward study [1], on the effects of combining cold exposure (CE) and exercise in humans.

I think it's safe to say that virtually every CRer, and in fact everyone seriously concerned with their health, engages in some form of exercise for its CVD-preventing, muscle-preserving and bone-building benefits. But some of us worry about the effects of the inflammation and the 'wear-and-tear' associated with exercise, particularly as we get older. And we aren't really interested in the getting big muscles, or raising our growth- and age-promoting testosterone & IGF-1 levels.

In addition, some of us (well, maybe just me☺) who nearly continuously expose ourselves to cold, combine it with nearly continuous very modest exercise for several reasons: to keep the blood circulating in the cold, to burn calories to maintain a net calorie deficit, and for all the wonderful benefits of CE described in my previous post and elsewhere in this thread.

But I've been a bit worried about the possible deleterious impact of combining CE and exercise. Might the extra physiological stress resulting from CE + exercise magnify the exercise-induced inflammation, and result in the accumulation of the kind of systemic damage that is the hallmark of aging - or more accurately, according to the SENS perspective, that is aging?

Fortunately, it looks like this is not the case - in fact quite the opposite. Study [1] found that relative to exercising in at room temperature, exercising at 0°C in shorts and a t-shirt (that's my kind of experiment!) resulted in the reduction of a wide range of inflammatory and growth promoting markers, including many interleukins and a bunch of others cytokines, several of which I've never heard of, including IFN-γ, Rantes, Eotaxin, IP-10, MIP-1β, MCP-1, VEGF, PDGF, and G-CSF [see Note 1].

Adding CE to exercise also blunted the exercise-induced increase in lymphocytes (white blood cells), which is also considered an inflammatory response. Reduced lymphocytes is a well known side effect of CR as well, although there is some reason to be concerned that this could negatively impact one's ability to fight off an established illness, as discussed here.

Adding CE to exercise also blunted the exercise-induced increase in testosterone and IGF-1. This too resembles the testosterone and IGF-1 lowering effects of long-term CR.

Interestingly, if they really tortured the subjects, by exposing them to a "pre-exercise low-intensity shivering protocol" (having them sit idle for 40-120min in 0°C in only shorts & t-shirt!) plus continued cold exposure during the subsequent exercise session, the combination reversed some of the beneficial reduction in inflammation and exacerbated the reduction in lymphocytes induced by exercising in the cold. It seems that bringing subjects to the point of shivering and then having them exercise in the cold was going too far to be beneficial. In a similar fashion, [2] found that chronic cold water exposure (without exercise, and to the point of inducing shivering) resulted in a modest elevation in markers of immune system activity, and a trend towards increased lymphocytes also. This result is corroborated by [3], which found that exposing men to 2 hours of a 5°C environment with a breeze blowing on them while wearing just shorts and socks boosted immune system activity, especially if the CE is preceded by exercise. So if you are concerned about suppressed immunity as a result of CR or some other malady, and you want to increase immune system activity, CE to the point of shivering, perhaps in association with exercise, might be a way to do it.

Here is the summary from the authors of [1]:

This study demonstrated that exercising in the cold can diminish the exercise-induced systemic inflammatory response seen in a thermoneutral environment. Nonetheless, prolonged cooling inducing shivering thermogenesis prior to exercise, may induce an immuno-stimulatory response following moderate intensity exercise. Performing exercise in cold environments can be a useful strategy in partially inhibiting the acute systemic inflammatory response from exercise but oppositely, additional body cooling may reverse this benefit.

In short, it appears the combination of non-shivering cold exposure + exercise is a win relative to exercise-alone when it comes to inflammation and pro-aging growth factors like testosterone and IGF-1. If you want to boost your immune system, cold exposure to the point of shivering looks to be an option.

--Dean

Note 1: The reduction of VEGF by cold exposure is interesting since elevated levels are implicated in a variety of cancers as well as macular degeneration, the latter of which is of particular concern for me.

Share this post

Link to post

Share on other sites

Here is an interesting one to tide people over while I finish up a big post on Speakman's work on cold exposure, BAT, metabolic rate & longevity.

In this study [1], researchers fed rats ad lib diets high in fat - 40% of calories, which not far from typical human consumption. They divided the rats into four groups, feeding them four different fats - olive oil (OO - MUFA-rich), sunflower oil (SO - PUFA-rich), palm oil (PO - SFA-rich), and beef tallow (BT - SFA-rich). No mention is made of the OO being "extra virgin", and it came from one of Spain's biggest olive oil companies (Koipe SA) so I think it's probably safe to assume it was run-of-the-mill olive oil rather than high-polyphenol EVOO. All the mice were housed at "normal" lab temperature (22 °C) for the duration of the experiment, which is chilly for rats.

After four weeks on each of the diets, all four groups had gained about the same amount of weight - about 50% of their initial body weight! Weight of BAT tissue wasn't significantly different between the groups.

What was interesting was the amount of gene expression of the three important uncoupling proteins, UCP-1, UCP-2 and UCP-3 in various tissues, including brown adipose tissue (BAT), white adipose tissue (WAT) and skeletal muscles. Here are graphs comparing UCP expression in BAT and muscle tissue for the four diets:

The letters (a, b) above the bars represent which groups were significantly different As you can see, olive oil significantly boosted UCP1, UCP2, and UCP3 messenger RNA expression in BAT, and UCP3 mRNA expression in muscle tissue relative to the other three fat sources.

In terms of actual UCP content in the various tissues on the various diets (as opposed to just messenger RNA expression shown above), only UCP2 was higher in BAT and UPC3 was higher in muscle tissue in the OO group relative to the other diets, particularly the SFA-rich diets:

As we'll see in my next post, both UCP2 and UCP3 are associated with increased longevity, so it is interesting to see them elevated in BAT and muscle tissue by OO.

Finally, while the OO diet increased total body oxygen consumption per gram of body weight (i.e. metabolic rate) relative to the other diets, oxygen consumption by BAT wasn't any different between groups. I suspect the rats weren't cold-stressed enough for BAT to majorly kick in, since they were feeding ad lib and gained over 50% of their initial body weight during the four weeks of the study (240g → ~370g), presumably mostly as highly insulating white fat.

The authors checked for a bunch of different possible causes for why OO might boost UCP gene expression including differential changes to circulating hormones, glucocorticoids, or insulin. Nope - none of them were significantly different across the diets. They suggest instead that it may be the oleic acid in OO being incorporated into cell membranes and increasing the responsiveness of cells to UPC-stimulating adrenaline:

The explanation of the effects of olive oil is not clear. It seems that these effects are not mediated by systemic metabolic changes, but rather may be related to a local effect produced by oleic acid on IBAT and gastrocnemius muscle. In our laboratory, we observed that, after a 4-wk olive oil feeding period, oleic acid concentrations were increased in the stored triacylglycerols (45) and also incorporated into the plasma membrane phospholipids (46) in both perirenal and subcutaneous WAT. Although not measured, it can be expected that the same would be true for the triacylglycerols stored in IBAT, skeletal muscle, and mitochondrial membranes. This potential increase in oleic acid might modify the response of IBAT and gastrocnemius muscle to adrenaline. Furthermore, the modifications in membrane phospholipids could lead to modifications of the membrane-receptor interactions of the transduction of the hormonal signal (23, 47–49).

So while olive oil didn't increase BAT activity per se, it did increase UCP gene expression and whole body oxygen consumption - perhap through UCP3-mediated mitochondrial uncoupling in skeletal muscles. If challenged by cooler temperatures and/or less food, I strongly suspect these increases in UCP gene activity would have also resulted in increased BAT activity. So I'm going to take the liberty of adding olive oil to the master list of BAT promoters.

Here is the latest full list of modifiable and [non-modifiable] factors associated with increased BAT quantity and/or activity:

Once again we see a food or behavior, in this case consumption of olive oil / MUFA, which is known to promote health / longevity also be a (likely) promoter of BAT activity - perhaps when coupled with cold exposure. You can judge for yourself whether all of these are coincidence or not...

P.S. I've edited the cold exposure and immunity post by adding this study (PMID: 10444630), which basically reinforces the conclusion that very serious cold exposure in humans (sitting wearing just shorts in 5°C with slightly breeze for 2 hours) elevates immune system activity.

P.P.S. I'm happy to report that this thread is #6 on the first page of results when you (or at least when I) do a Google search for "cold exposure longevity".

They used one of these Blanketrol III circulating water cooler machines from Cincinnati Sub-Zero for rapidly cooling the research subjects:

We conclude that cold stimulation in humans increases BATassociated EE and whole-body EE; however, BAT is minor contributor to this whole-body cold-induced thermogenesis, while deeper centrally located neck muscles, along with the pectoral muscles are among the major contributors to thermogenesis. Moreover, in BAT, both during RT and cold stress, oxygen consumption is interlinked with the circulatory uptake of NEFA

I'd like to know more about this "deep neck and pectoral" muscle contribution to cold induced thermogenesis. Not sure if this has anything to do with shivering? Or something else. Study sounded like they cooled down to the shiver point then raised the temps presumably so there was no more shivering. This study also notes the link between BAT activity and non-esterified fatty acid uptake.

Edited March 24, 2016 by Gordo

Share this post

Link to post

Share on other sites

We conclude that cold stimulation in humans increases BAT-associated EE and whole-body EE; however, BAT is minor contributor to this whole-body cold-induced thermogenesis, while deeper centrally located neck muscles, along with the pectoral muscles are among the major contributors to thermogenesis.

Link to post

Share on other sites

But seriously, I had been wondering at first if test subjects were flexing muscles to stay warm kind of like people in this thread reported having to keep moving when cold? But it appears that this BAT to "nearest muscle" interaction is involuntary, and in fact the muscle based thermogenesis was directly correlated with BAT quantity in test subjects. So there may be an interesting relationship between BAT and the muscle that surrounds it. This suggests that building up neck and pec muscles might be beneficial if not essential to one's personal "BAT building regimen". I've been doing short duration HIIT workouts for the last 2 years with a particular focus on pull ups and hanging crunches, which has resulted in pretty significant pectoral and neck muscle development, so perhaps that is one more thing contributing to my apparently easy ability to activate BAT/cold induced thermogenesis. I only work out for 5 minutes or less per day and eat a superfood rich, low protein plant based whole food diet (with no protein supplementation) which seems to be adequate:

This may also partially explain why cold induced thermogenesis is so low or non-existent in severely CR'ed (anorexic) subjects with no muscle mass (and may also have something to do with a similar lack of cold induced thermogenesis in obese subjects?).

Edited March 25, 2016 by Gordo

Share this post

Link to post

Share on other sites

Thanks for the pointer to this interesting, and it possibly quite important study (PMID 26993316). It has sent me down a two-day (very interesting and productive!) rabbit hole investigating muscle-related thermogenesis. I'm in the midst of a post about it now (putting my Speakman post on hold...).

But two quick responses to points you two have brought up. First, regarding the suggestion that the heat production by muscles is either voluntary (i.e. flexing/clinching) or involuntary but nonetheless contraction-related (i.e. shivering). It appears from the evidence that I'll present that non-shivering thermogenesis (NST) is the primary way muscles generate heat unless it's really cold and you start shivering - which the researchers took explicit steps in PMID 26993316 to avoid. I'll talk about the mechanism of NST in muscle tissue and its significance in my upcoming post.

Second, the reason PMID 26993316 only looked at the upper body Gordo is that the scanner they used has a narrow field of view. They wanted to look at BAT, so they pointed it at the part of the body where BAT is concentrated, and so only imaged muscles in the chest and neck region. BAT may indeed be signally nearby muscles to generate heat, but this study doesn't help to determine that, since it only looked at muscles that are in the vicinity of BAT pockets.

--Dean

Share this post

Link to post

Share on other sites

In this post I discussed study [1], which found that cold exposure not only increases BAT in mice so they can burn more calories to stay warm, but it also shifts their gut microbiome ... As I discussed in this post, I appear to have 2x more Firmicutes relative to Bacteroidetes in my latest tests, and be enriched in Firmicutes relative to the general population. ... these shifts (i.e. an ↑ in Firmicutes and ↓ in Verrucomicrobia) are in the same direction as the cold-exposed mice. Interestingly, as of the date of those two 2015 tests, I wasn't yet intentionally engaged in my cold exposure experiments....Does anyone else, cold-adapted or not, have uBiome data on Firmicutes and Verrucomicrobia they'd be willing to share?

My ubiome numbers from Nov. 2013 when I was surfing in cold water 1hr/day were Firmicutes 19.6% above the reference group and Verrucomicrobia 1/3rd of the reference group.

Share this post

Link to post

Share on other sites

Dean -- thank you for putting together all that research and thoughtful analysis - great information. This thread is now the #2 web result link that comes up from a search on "cold exposure longevity" from BOTH bing.com and google.

I do think cold exposure has likely been a confounding factor in past DR/CR studies in rodents. I have even contacted the researcher doing this ongoing study of DR in mice which may be of interest to people here. He was nice enough to respond, but didn't seem to think CE was a factor. Professor Arlan Richardson says:

Hollozy’s study showed that cold exposure did not change lifespan and in a study done at San Antonio (Ikeno et al.) they showed housing mice 1 vs 4 had the same lifespan whether fed AL or DR (in fact the DR mice group housed lived a little, but not significant, longer)

I have not tracked down those references.

I have been continuing my personal observations/anecdotes with CE and blood glucose and have been seeing very positive results so far. I'll post more details after doing more rigorous experiments with multiple data points. I feel like CE opens the door to a LOT of foods with well documented health benefits that happen to also be higher glycemic index foods including most whole grains and rice, honey, maple syrup, most fruit especially things like apples, bananas, etc. Apples are of interest to me since I grow them, and their (organic) skins especially are top superfoods with many beneficial properties. Maple syrup is another personal interest not only because I have a maple grove behind my house that I tap for personal use, but because pure maple syrup has been shown in various research to have anti-cancer, anti-inflammatory, and anti-diabetic properties (oddly enough), plus loads of phytonutrients/compounds not found in any other foods. More recently it has even been shown to prevent the misfolding of brain cell proteins (related to aging/alzheimers/dimentia) See:http://www.sciencecodex.com/uri_scientist_discovers_54...http://www.lifeextension.com/news/lefdailynews...http://www.sciencedaily.com/rele.../2010/03/100321182924.htmhttp://www.medicaldaily.com/maple-syrup-alzheimers-disease-brain-health-377846

Many of the same types of health benefits have been associated with raw pure honey as well.

But how can you eat all these great, health promoting foods without sending your blood sugar soaring? Some longevists take metformin for this purpose, but that always seemed like a bad idea to me, and has many side effects and drawbacks. Cold exposure is a better, completely natural, alternative.

Another personal anecdote - last night I had a huge bowl of porridge consisting of barley and oats (half cup dry of each) plus almond milk, tart cherries (Aldi's dark morello), high GI dried cranberries, walnuts, blueberries, flax, chia, lemon, a tablespoon of maple syrup and a tablespoon of buckwheat honey. I think I am going to make this my reference meal for more rigorous future experiments. I will more carefully measure/weigh everything next time and plug it into software for analysis. Anyway this is a meal that I'd expect to send blood sugar soaring. Unfortunately I again only took one postprandial measurement after CE so that doesn't say a whole lot, but I was pleased to see a 77 blood glucose reading (I have taken measurements with the same device just before and after official lab testing and it was spot on).

Share this post

Link to post

Share on other sites

I really must thank you for presenting challenges to the "Cold Exposure Hypothesis" like this new study [1] (PMID 26993316). In short, it used what appears to be a relatively new imaging technique to assess the metabolic activity of human upper body BAT and muscle tissue during cold exposure. In agreement with many previous studies we've discussed, it found that BAT activity was increased by cold exposure in humans (by 50%), and that BAT mass and BAT activity correlated with increased cold-induced thermogenesis and energy expenditure.

But, in fascinating twist, it found that the actual contribution of BAT activity to thermogenesis and increased energy expenditure (as measured directly by oxygen consumption) in response to cold appeared to be quite modest in humans - on the order of 10 kcal out of the total increase of around 350 kcal per day. Further, they found that the upper body skeletal muscles (including Michael's pectoral muscles - nice video!) contributed far more to cold-induced thermogenesis than did BAT. This finding would seem to be corroborated by [2], which found that even in people with a relatively large amount of BAT, the actual contribution of BAT activity to cold-induced thermogenesis is small compared to the total size of the thermogenic response. The imaging technique the researchers employed in both these studies is pretty new and novel, so it's hard to say just how definitive these results are concerning the magnitude of BAT's role in human thermogenesis. But they can't be easily dismissed, since previous human studies have focused on the correlation of BAT mass and BAT activity with overall cold-induced thermogenesis, without showing that BAT was directly responsible for most or all of the observed increase in energy expenditure.

The authors of [1] are uncertain just how the muscles are generating heat. Subjects were told to relax their muscles and were lying down in a comfortable position, so voluntary muscle flexion (as so amusingly demonstrate in Michael's and Gordo videos) was unlikely, although can't be ruled out. Or possibly they could have been clenching their muscles involuntarily to cope with the cold. This too can't be ruled out, but seems unlikely since you would think subjects would have reported it. Similarly, they could been shivering imperceptibly, and generating heat that way. But the researchers took steps to prevent shivering by decreasing the severity of the cold when they or the subjects detected shivering.

Alternatively, the authors of [1] suggest that the increased energy expenditure in muscle cells could result from non-shivering thermogenesis (which I'll refer to as smNST for "skeletal muscle non-shivering thermogenesis"), and that this could result from increased expression of uncoupling proteins in muscles, like what occurs with UCP-1 in BAT. In particular, there is pretty good evidence that short-term (24h) cold exposure upregulates UPC-3 in mitochondria of rat muscle cells, and a lower mitochondrial membrane potential, increasing heat production and also potentially lowering ROS production [4]. But [5] and others have found that UCP-3 expression in muscle cells is transiently elevated by cold, but returns to baseline after chronic cold exposure (15 days). Or smNST could be increased by expression of UCP-2 in muscles as a result of cold exposure, which was observed in mice [21] and humans [20]. Alternatively, study [5] suggests that free fatty acids rather than uncoupling proteins, may be the cause of increased proton leakage and heat production in skeletal muscle mitochondria.

Tantalizingly, quite a number of studies suggest mitochondrial uncoupling might be associated with increased longevity. Studies [22][23] found that increased UCP-2 expression results in increased longevity in rodents, and this may be associated with body temperature regulation [25], although this result is controversial [24]. Similarly suggestive, [26] found that mice chronically treated with a mild mitochondrial uncoupling agent that works independently of the UCPs (a chemical called 2,4-dinitrophenol or DPN) resulted in mice who ate more, weighed less, had lower measures of fasting glucose, triglycerides, insulin and oxidative damage to DNA, and most importantly, lived longer than controls - mean and maximum (oldest 10%) lifespan were extended by 7% and ~11%, respectively. And in this post, I talk about the evidence that increased expression of UCP proteins is associated with greater human longevity.

Regardless of the mechanism of mitochondrial uncoupling, and regardless of whether uncoupling is occurring in BAT, skeletal muscle, other organs, or some combination, it appears to be happening as a result of cold exposure, and have the potential to be health and lifespan promoting. The importance of mitochondrial uncoupling for thermogenesis in humans in response to cold exposure is demonstrated by [19], in which resting metabolic rate was increased by 76kcal/day as a result of mild cold exposure (80 hours @ 16 °F), and about half of that increase appeared to result from mitochondrial uncoupling.

But there is another, very recently discovered (only since 2012 - more recently than human BAT!) mechanism for mammalian smNST that appears quite significant. In fact I'm surprised the authors of [1] didn't mention it.

But before getting into the specific mechanism, a word about the significance of smNST for cold adaptation in humans and rodents. We've (mostly I've ☺) focused mainly on BAT-thermogenesis in response to cold. But apparently even in rodents, smNST is a very important response to cold adaptation, on par with BAT. Study [3] found that mice whose BAT had been removed and who were given a drug to prevent shivering, were still able to maintain body temperature via smNST. And no, they weren't clenching their muscles or increasing their activity level to generate heat. Instead, they were generating heat via smNST using a pathway I'll describe in detail below. How did the authors know this? Mice with a knockout mutation for an important protein involved in this new form of smNST, whose BAT had been removed and shivering inhibited, were unable to maintain their body temperature in response to cold, in contrast with WT mice without the knockout mutation given the same treatment. In fact, the poor mice lacking the ability for smNST either died or had to be removed from the cold conditions. So the point is that it's not just humans where smNST can play a very important role in cold-induced thermogenesis, but rodents as well.

The protein alluded to above that facilitates smNST is called Sarcolipin. I found the way it works to be very interesting, but required me to bone up on my muscle biochemistry. Bear with me, or skip this and the next two paragraph if you aren't interested in the mechanism, only the result. This is highly simplified, but skeletal muscles contract as a result of calcium ions (Ca++) flooding the muscle cell cytoplasm, causing a sequence of reactions that result in muscle fiber shortening. Where does the flood of Ca++ ions come from? It is stored up in a cellular organelle called the sacroplasmic reticulum (SR), which is related to, but differs in structure and function from, the endoplasmic reticulum that people have probably heard of. The SR's primary role in muscle cells is to serve as the reservoir for Ca++ ions. How do the Ca++ ions get into the SR? Through active transport by a pump called SERCA (I'll spare you what SERCA stands for). Basically when ATP and two Ca++ ions bind to the SERCA pump on the outside of the SR membrane, the ATP undergoes hydrolysis (conversion to ADP) and the two Ca++ ions get transported through the SERCA complex across the SR membrane to the interior of the SR. This creates a strong gradient, with a much greater concentration of Ca++ ions on the inside of the SR than on the outside. When a motor neuron signals to the muscle cell that it's time to contract, what basically happens is that the SR gets the message and suddenly releases all it's built-up Ca++ ions, causing the muscle fibers to contract. And that, in a very simplified nutshell, is how muscles contract.

OK you might be saying, but what does the fact that SERCA pumps Ca++ ions to enable our muscles to contract and relax have to do with thermogenesis? Well, because this calcium ion pumping is a very energy hungry, heat generating process. According to [15] (a great review of all aspects of mammalian thermogenesis by pioneers of the research I'm describing), SERCA activity comprises approximately 42% to 48% of resting metabolic rate in slow and fast-twitch muscles. Even more significant, SERCA activity can account for approximately 12% to 15% of whole-body resting energy expenditure and approximately 15% to 20% of total daily energy expenditure [15]. So unlike BAT which varies dramatically between species, SERCA is known to play a big role in metabolism and energy expenditure across all mammalian species.

So you might ask, where does sarcolipin come in to generate smNST out of all this? Sarcolipin binds to the SERCA pump, causing it to go through the motion of hydrolyzing ATP to ADP like usual, but instead of actually transferring it's captured Ca++ ions into the interior of the SR, the SERCA pump simple releases its Ca++ ions back into the muscle cell cytoplasm. This is known as 'futile cycling' - because ATP is being reduced to ADP, thereby expending energy and generating heat, but no work is getting done (i.e. no Ca++ ions are getting transported from the cytoplasm across the SR membrane to the SR interior). In fact, more heat is generated per ATP molecule hydrolyzed when sarcolipin causes SERCA to drop its Ca++ ions early and not actually pump them across the SR membrane. And because the SERCA pump is trying to increase the reservoir of Ca++ ions inside the SR so the muscle can get ready to fire again, it will keep trying (futilely) to pump Ca++ ions, expending more energy and generating more heat.

So how significant is this sarcolipin-mediated smNST for burning calories and generating heat in real life? Study [3] found that sarcolipin knockout mice were prone to diet-induced obesity compared with normal mice. Even more dramatically, study [6] studied mice in which sarcolipin was either knocked out or overexpressed. They found that when the knockout and overexpressing mice were fed the same amount of food, the knockout mice gained 22% of their body weight (as fat) and the sarcolipin overexpressing mice lost 23% of their body weight. In the end the sarcolipin-knockout mice weighted ~50% more than the sarcolipin-overexpressing mice, while eating the exact same amount of food. When fed ad lib, the sarcolipin overexpressing mice ate ~37% more food, but gained ~66% less weight and weighed half as much as the obese sarcolipin knockout mice; these effects were not a result of increased physical activity - the researchers ruled that out. Here is the dramatic difference in weight trajectories for the sarcolipin knockout mice (Sln-/-), sarcolipin overexpressing mice (SlnOE) and wild-type (WT) mice when fed a high fat diet ad lib from [6]:

So in rodents at least, sarcolipin-induced smNST can have a really big effect on calorie expenditure and body weight, even at the thermoneutral temperature employed in this study (29 °C) to suppress BAT thermogenesis that might have masked the effects of sarcolipin-induced smNST. Interestingly, the tendency of sarcolipin-knockout mice to become obese when fed ad lib occurred despite a "comparable increase in BAT mass and UCP-1 content to that of wild-type mice" and despite eating the same amount of food and being no less active than wild-type mice. "This is in stark contrast to UCP-1 ablation, which is not obesogenic under standard animal housing conditions" [15]. In other words, even in rodents where BAT is prevalent and important for thermogenesis, sarcolipin-induced smNST appears more important for maintaining energy balance. As the authors of [15] put it:

And it's not just in these mutant mice that variations in sarcolipin are associated with changes in energy balance. In wild-type mice, sarcolipin expression increases by a factor of 3-5x as a result of high fat feeding [15]. Unlike BAT, which kicks in mostly as a result of cold exposure and only modestly to compensate for excess calories, sarcolipin (SLN in this diagram) can have important effects on metabolism across a range of energy demands, as illustrated in this diagram from [15]:

This "gaining less weight while eating more food" as a result of extra sarcolipin brings to mind the "constitutionally lean" (CL) women from PMID 23393181 discussed in this post. Like the sarcolipin-overexpressing mice, the CL women had a naturally higher metabolic rate and were resistant to weight gain when overfed. And both the obesity-resistant, sarcolipin-overexpressing mice in [6] and the the CL women in PMID 23393181 had lower respiratory quotient, suggesting increased burning of fat relative to carbohydrates. The CL women were also shown to be the only ones with significant BAT, while anorexics, recovered anorexics, or normal-weight women who had none. But could their extra BAT be just part the metabolic program that enables them to stay slim? In particular, could it be that their muscles were also burning more calories, perhaps through this sarcolipin-induced smNST? And could it be what's going on in the muscles of the cold-exposed men in the study that triggered this discussion (PMID 26993316), whose muscles were burning a lot of calories apparently without shivering or flexing/contracting? It seems like a real possibility to me, and to many of the authors of the recent flurry of sarcolipin papers, such as this quote from [15]:

Although active BAT now has been confirmed in adult humans in small quantity, it is not detected in all individuals (ref) and only accounts for a small fraction of energy expenditure during physiological stress (ref). Thus, an alternative means of adaptive thermogenesis may lie within skeletal muscle of certain individuals and may involve [sarcolipin], considering it is expressed abundantly in human skeletal muscle (ref).

and this one from the model proposed in [16]:

In adult non-hibernating mammals, including humans, where UCP1 and BAT are limited, [sarcolipin] is the dominant source of thermogenesis. By contrast, in species where UCP1 and BAT are abundant, the contribution of [sarcolipin] to thermogenesis is secondary to that of BAT.

It seems to me like sarcolipin may play an important role in human thermogenesis. But unfortunately there appears to be little research so far on sarcolipin-induced smNST in humans. Sarcolipin has long been known to be an important protein for regulating heart muscle tissue in humans, and sarcolipin has recently been shown to be abundant in human skeletal muscle fibers as well, in fact in much more abundance than in the muscles of rodents [16], suggesting it may be playing the thermogenic role in humans that BAT/UCP-1 plays in small mammals [14][16] - unless the rodents have their BAT/UCP-1 knocked out, in which case they apparently rely on upregulation of sarcolipin-induced smNST to cope with thermal stress. But definitive evidence of sarcolipin's involvement in human thermogenesis does not appear to yet be available. About all I could find was [7] which found sarcolipin-induced respiration (i.e. futile cycling & heat generation) was seriously reduced in morbidly obese humans (BMI around 50!) compared with lean humans.

But does sarcolipin-mediated smNST really kick in in response to cold exposure? As we saw in [3], it can be quite significant in rodents, on par with the amount of heat produced by BAT, and the difference between life and death for cold-exposed BAT-less rodents lacking the gene to produce sarcolipin. Study [11] found cold-reared mice (4 °C) expressed between 3x and 10x as much sarcolipin (depending on which muscle was tested) than did warm-reared mice. Even more impressive is this study [8] in rabbits, which don't have any BAT but which are quite good at cold adaptation. Researchers kept rabbits either at normal room temperature or 4 °C for 10 days. The cold rabbits maintained normal body temperature, ate about 15% more, and gained 66% less weight as the warm rabbits when fed ad lib. So the cold rabbits were expending a lot more energy, but they didn't have BAT and weren't shivering or running around to keep warm, so how were they doing it? Apparently via futile cycling of the SERCA calcium channel discussed above. Muscle cells isolated from the cold-adapted rabbits showed a dramatic increase SERCA pump concentration and activity. As a result, the muscles of the cold-adapted rabbits produced twice as heat as the warm rabbits. Interestingly, this paper was from 2008 (4 years before the thermogenic role of sarcolipin was discovered), so they weren't quite sure how SERCA activity was resulting in so much heat generation. We now know the mechanism is likely to be the uncoupling of ATP hydrolysis from the transport of Ca++ ions - i.e. futile cycling in the SERCA pumps induced by sarcolipin.

But will upregulation of sarcolipin and SERCA pumps to support smNST compromise muscle performance? In other words, if sarcolipin is attached to all the SERCA pumps and so causing futile cycling, will the pumps be unable to restore the Ca++ ion gradient to allow effective muscle performance? To find out, study [9] looked at muscle performance in mice after 5 weeks of cold exposure (4 °C), compared to muscles from mice housed at 24 °C. They chose a muscle that did not participate in shivering to avoid the impact of shivering on their results. Consistent with the studies above, cold exposed mice showed 4x as much Ca++ ion leakage from the sarcoplasmic reticulum (SR) as the warm mice - a sign that futile cycling of the SERCA calcium pumps in the SR, almost certainly induced by sarcolipin (although this was from 2010 before sarcolipin's role was discovered), was playing a big role in keeping the cold-adapted mice warm. So what about muscle performance - were the cold-adapted mice weaklings because of all that Ca++ ion leakage? Nope - quite the opposite in fact, as shown in the figure below:

As you can see, the muscle fibers from the cold-adapted mice (right panel) didn't get fatigued nearly as quickly as the fibers from the warm-adapted mice (left panel). The authors conclude (my emphasis):

Similarly, and in support of the hypothesis that the cold-induced muscle performance improvements seen in [9] were indeed mediated by sarcolipin, study [10] found that muscles were more fatigue resistant in mice genetically altered to overexpress sarcolipin than the muscles of wild-type mice. Those same sarcolipin-upregulated mice were able to run 16% farther than WT-mice before exhaustion in an progressively more difficult treadmill endurance test.

Based on [9] and [10] together, it appears cold-exposure boosted mitochondrial size and/or number ("increased mitochondrial content") and enabled the SR in muscle fibers to release 40% more calcium ions in response to stimulation ("higher tetanic [Ca2+]i") enabling stronger & more prolonged contraction during repeated stimulation, the same way endurance exercise improves muscle fiber performance. I know Michael will probably be skeptical about the claim about increased mitochondria. But data from [6] shows pretty definitively that the over or underexpression of sarcolipin (via genetic manipulation) results in large changes in both the number of mitochondria and the total amount of mitochondrial DNA (relatively to nuclear DNA) in two different mice muscles, the Tibialis Anterior (TA) and Extensor Digitorum Longus (EDL). Here is the graph comparing the mitochondria count and DNA content in sarcolipin knockout mice (Sln-/-), sarcolipin overexpressing mice (SlnOE) and wild-type (WT) mice:

How cool is that? What this strongly suggests is that cold exposure can not only burn calories via non-shivering thermogenesis in skeletal muscles, it also conditions muscles to perform better in the same way endurance exercise conditions them. In other words, you can burn calories and get in better shape via cold exposure without the wear and tear, or the pain of exercise. As a bonus, increased mitochondrial content in muscle cells may increase longevity. For example, CR boosts the number of mitochondria in skeletal muscles of rats [17] and humans in the CALERIE study [18], and this is thought to be a means by which CR reduces oxidative damage and possibly how it improves longevity i.e. spreading the workload between more mitochondria is thought to reduce free-radical damage.

Thanks for anyone who've stuck with me this far (and even those who've skipped to the bottom to see the upshot). I'll try to summarize what all this likely means:

It looks like BAT may not be the only game in town when it comes to cold-induced thermogenesis. In particular, non-shivering thermogenesis in skeletal muscles looks like it may be quite important, particularly in large mammals like humans that don't have as much BAT as small mammals like rodents.

Uncoupling proteins might be part of the mechanism by which humans generate extra heat in response to cold.

But it seems more likely that increased cold-induced thermogenesis results from an increase in the expression of the protein sarcolipin, which generates heat and expends more energy by uncoupling calcium ion pumping in skeletal muscle sarcoplasmic reticulum.

Sarcolipin-induced changes in calcium ion exchange and mitochondria biogenesis appears to improve muscle performance (reduce fatigue) in the same way endurance exercise improves muscle performance, and may reduce oxidative damage in muscle cells as well, perhaps improving health and longevity.

While sarcolipin's role in human thermogenesis is still speculative, I predict we'll see a lot of study of this very soon, given its therapeutic potential for treatment of obesity. Unlike BAT (which people have been excited about for obesity avoidance for a while now), which is minimal in most people and requires generating lots of new cells/tissue to have an effect, sarcolipin is just a protein, the action of which might be mimicked or bolstered directly via drugs rather than cold exposure, making it a much more promising avenue than boosting BAT or BAT activity for pharmaceutical companies to explore as a way to treat obesity.

Finally, I can hear the critics & skeptics saying:

But Dean, you've been harping on BAT all along as the likely mechanism by which cold exposure is beneficial. Are you now changing you tune? What about all those other studies you've been posting about the importance of BAT?"

First I'll note that my "Cold Exposure Hypothesis" is that cold exposure is beneficial, and BAT may be one mechanism by which those benefits are generated. The fact that there may be other ways as well, like sarcolipin-induced non-shivering thermogenesis in skeletal muscles, provides additional evidence to support, rather than refute, my hypothesis. Moreover, all this stuff about cold exposure influencing muscle thermogenesis doesn't negate the BAT-related evidence I've presented previously. It's clear that BAT is elevated by cold exposure in humans, does generate heat and burn calories, and is likely to be associated with beneficial health & longevity effects. Even if BAT's direct contribution to energy expenditure is relatively modest in humans, it is very actively involved in endocrine signalling, and may even upregulate glucose and/or fatty acid metabolism in skeletal muscles via FGF-21 or adiponectin release as suggested by [12] and [13]. So BAT and skeletal muscles appear to work synergistically to adapt to the stress of cold exposure.

Finally, skeletal muscles make up around 40% of a healthy person's body mass and the maintenance of muscle function is critically important for healthy aging. The fact that cold exposure appears to impact muscle directly, and in a way similar to endurance exercise and calorie restriction, shows yet another pathway by which cold exposure may improve health and longevity. This new mechanism, and particularly its apparent effects on muscle mitochondria, might even expand Michael's imagination about the way cold exposure could beneficially impact aging.

--Dean

----------

[1] Eur J Nucl Med Mol Imaging. 2016 Mar 19. [Epub ahead of print]

Human brown adipose tissue [(15)O]O2 PET imaging in the presence and absence of

Author information:(1)Department of Bioenergetics, AN Belozersky Institute of Physico-ChemicalBiology, Moscow State University, Russia.

The mechanism of thermoregulatory uncoupling of respiration and phosphorylationin skeletal muscles has been studied. It is found that 24 h cold exposure resultsin (i) a 3-fold increase in the amount of UCP3 protein in rat skeletal musclemitochondria, and (ii) pronounced lowering of the membrane potential in isolatedrat or mouse skeletal muscle mitochondria. The decrease in membrane potential isreversed by adding bovine serum albumin. Cold exposure is also found to sensitizethe membrane potential to the uncoupling action of added fatty acid (laurate).After laurate addition, the recoupling effects of GDP and carboxyatractylatedecrease whereas that of albumin increases in mitochondria from cold-treated ratsor mice. Changes similar to those induced by cold can be initiated by the in vivoaddition of thyroxine. Cold exposure does not affect energy coupling in livermitochondria. The possible involvement of UCP3 isoforms in nucleotide-sensitiveand -insensitive uncoupling is discussed.

Mammals exposed to a cold environment initially generate heat by repetitivemuscle activity (shivering). Shivering is successively replaced by therecruitment of uncoupling protein-1 (UCP1)-dependent heat production in brownadipose tissue. Interestingly, adaptations observed in skeletal muscles ofcold-exposed animals are similar to those observed with endurance training. Wehypothesized that increased myoplasmic free [Ca2+] ([Ca2+]i) is important forthese adaptations. To test this hypothesis, experiments were performed on flexordigitorum brevis (FDB) muscles, which do not participate in the shiveringresponse, of adult wild-type (WT) and UCP1-ablated (UCP1-KO) mice kept either atroom temperature (24°C) or cold-acclimated (4°C) for 4-5 weeks. [Ca2+]i (measuredwith indo-1) and force were measured under control conditions and during fatigueinduced by repeated tetanic stimulation in intact single fibres. The results showno differences between fibres from WT and UCP1-KO mice. However, muscle fibresfrom cold-acclimated mice showed significant increases in basal [Ca2+]i (∼50%),tetanic [Ca2+]i (∼40%), and sarcoplasmic reticulum (SR) Ca2+ leak (∼fourfold) ascompared to fibres from room-temperature mice. Muscles of cold-acclimated miceshowed increased expression of peroxisome proliferator-activated receptor-γcoactivator-1α (PGC-1α) and increased citrate synthase activity (reflectingincreased mitochondrial content). Fibres of cold-acclimated mice were morefatigue resistant with higher tetanic [Ca2+]i and less force loss duringfatiguing stimulation. In conclusion, cold exposure induces changes in FDBmuscles similar to those observed with endurance training and we propose thatincreased [Ca2+]i is a key factor underlying these adaptations.

Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase inskeletal muscle. Recent studies using SLN-null mice have identified SLN as a keyplayer in muscle thermogenesis and metabolism. In this study, we exploited a SLNoverexpression (Sln(OE)) mouse model to determine whether increased SLN levelaffected muscle contractile properties, exercise capacity/fatigue, and metabolicrate in whole animals and isolated muscle. We found that Sln(OE) mice are moreresistant to fatigue and can run significantly longer distances than wild-type(WT). Studies with isolated extensor digitorum longus (EDL) muscles showed thatSln(OE) EDL produced higher twitch force than WT. The force-frequency curves werenot different between WT and Sln(OE) EDLs, but at lower frequencies thepyruvate-induced potentiation of force was significantly higher in Sln(OE) EDL.SLN overexpression did not alter the twitch and force-frequency curve in isolatedsoleus muscle. However, during a 10-min fatigue protocol, both EDL and soleusfrom Sln(OE) mice fatigued significantly less than WT muscles. Interestingly,Sln(OE) muscles showed higher carnitine palmitoyl transferase-1 proteinexpression, which could enhance fatty acid metabolism. In addition, lactatedehydrogenase expression was higher in Sln(OE) EDL, suggesting increasedglycolytic capacity. We also found an increase in store-operated calcium entry(SOCE) in isolated flexor digitorum brevis fibers of Sln(OE) compared with WTmice. These data allow us to conclude that increased SLN expression improvesskeletal muscle performance during prolonged muscle activity by increasing SOCEand muscle energetics.

White adipose tissue is recognized as both a site of energy storage and anendocrine organ that produces a myriad of endocrine factors called adipokines.Brown adipose tissue (BAT) is the main site of nonshivering thermogenesis inmammals. The amount and activity of brown adipocytes are associated withprotection against obesity and associated metabolic alterations. These effects ofBAT are traditionally attributed to its capacity for the oxidation of fatty acidsand glucose to sustain thermogenesis. However, recent data suggest that thebeneficial effects of BAT could involve a previously unrecognized endocrine rolethrough the release of endocrine factors. Several signaling molecules withendocrine properties have been found to be released by brown fat, especiallyunder conditions of thermogenic activation. Moreover, experimental BATtransplantation has been shown to improve glucose tolerance and insulinsensitivity mainly by influencing hepatic and cardiac function. It has beenproposed that these effects are due to the release of endocrine factors by brownfat, such as insulin-like growth factor I, interleukin-6, or fibroblast growthfactor-21. Further research is needed to determine whether brown fat plays anendocrine role and, if so, to comprehensively identify which endocrine factorsare released by BAT. Such research may reveal novel clues for the observedassociation between brown adipocyte activity and a healthy metabolic profile, andit could also enlarge a current view of potential therapeutic tools for obesityand associated metabolic diseases.

Author information:(1)Department of Internal Medicine, Graduate School of Medicine, University ofTokyo, Tokyo, Japan.

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energyhomeostasis and glucose and lipid metabolism. However, the signaling pathwaysthat mediate the metabolic effects of Ad remain poorly identified. Here we showthat phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK)are stimulated with globular and full-length Ad in skeletal muscle and only withfull-length Ad in the liver. In parallel with its activation of AMPK, Adstimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acidoxidation, glucose uptake and lactate production in myocytes, phosphorylation ofACC and reduction of molecules involved in gluconeogenesis in the liver, andreduction of glucose levels in vivo. Blocking AMPK activation bydominant-negative mutant inhibits each of these effects, indicating thatstimulation of glucose utilization and fatty-acid oxidation by Ad occurs throughactivation of AMPK. Our data may provide a novel paradigm that anadipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directlyregulating glucose metabolism and insulin sensitivity in vitro and in vivo.

Thermogenesis is one of the most important homeostatic mechanisms that evolvedduring vertebrate evolution. Despite its importance for the survival of theorganism, the mechanistic details behind various thermogenic processes remainincompletely understood. Although heat production from muscle has long beenrecognized as a thermogenic mechanism, whether muscle can produce heatindependently of contraction remains controversial. Studies in birds and mammalssuggest that skeletal muscle can be an important site of non-shiveringthermogenesis (NST) and can be recruited during cold adaptation, althoughunequivocal evidence is lacking. Much research on thermogenesis during the lasttwo decades has been focused on brown adipose tissue (BAT). These studies clearlyimplicate BAT as an important site of NST in mammals, in particular in newbornsand rodents. However, BAT is either absent, as in birds and pigs, or is only aminor component, as in adult large mammals including humans, bringing intoquestion the BAT-centric view of thermogenesis. This review focuses on theevolution and emergence of various thermogenic mechanisms in vertebrates fromfish to man. A careful analysis of the existing data reveals that muscle was theearliest facultative thermogenic organ to emerge in vertebrates, long before theappearance of BAT in eutherian mammals. Additionally, these studies suggest thatmuscle-based thermogenesis is the dominant mechanism of heat production in manyspecies including birds, marsupials, and certain mammals where BAT-mediatedthermogenesis is absent or limited. We discuss the relevance of our recentfindings showing that uncoupling of sarco(endo)plasmic reticulum Ca(2+)-ATPase(SERCA) by sarcolipin (SLN), resulting in futile cycling and increased heatproduction, could be the basis for NST in skeletal muscle. The overall goal ofthis review is to highlight the role of skeletal muscle as a thermogenic organand provide a balanced view of thermogenesis in vertebrates.

BACKGROUND: Mild cold exposure and overfeeding are known to elevate energyexpenditure in mammals, including humans. This process is called adaptivethermogenesis. In small animals, adaptive thermogenesis is mainly caused bymitochondrial uncoupling in brown adipose tissue and regulated via thesympathetic nervous system. In humans, skeletal muscle is a candidate tissue,known to account for a large part of the epinephrine-induced increase in energyexpenditure. However, mitochondrial uncoupling in skeletal muscle has notextensively been studied in relation to adaptive thermogenesis in humans.Therefore we hypothesized that cold-induced adaptive thermogenesis in humans isaccompanied by an increase in mitochondrial uncoupling in skeletal muscle.METHODOLOGY/PRINCIPAL FINDINGS: The metabolic response to mild cold exposure in11 lean, male subjects was measured in a respiration chamber at baseline and mildcold exposure. Skeletal muscle mitochondrial uncoupling (state 4) was measured inmuscle biopsies taken at the end of the respiration chamber stays. Mild coldexposure caused a significant increase in 24h energy expenditure of 2.8% (0.32MJ/day, range of -0.21 to 1.66 MJ/day, p<0.05). The individual increases inenergy expenditure correlated to state 4 respiration (p<0.02, R(2) = 0.50).CONCLUSIONS/SIGNIFICANCE: This study for the first time shows that in humans,skeletal muscle has the intrinsic capacity for cold induced adaptivethermogenesis via mitochondrial uncoupling under physiological conditions. Thisopens possibilities for mitochondrial uncoupling as an alternative therapeutictarget in the treatment of obesity.

Author information:(1)Department of Medical Biochemistry, Faculty of Medicine, University of Geneva,Switzerland. Olivier.Boss@medecine.unige.ch

The control of uncoupling protein-2 (UCP2) mRNA expression in rat brown adiposetissue (BAT), heart and skeletal muscles was examined. Cold exposure (48 h)increased UCP2 mRNA in BAT, heart and soleus muscle by 2.4-, 4.3- and 2.6-fold,respectively. Fasting (48 h) had no effect on UCP2 mRNA expression neither in BATnor in heart, but markedly increased it in skeletal muscles. While theupregulation of UCP2 mRNA in response to cold exposure is in line with a putativeuncoupling role for this protein in thermoregulatory thermogenesis, theunexpected upregulation of UCP2 in skeletal muscles in response to fasting seemsinconsistent with its role as an uncoupling protein involved in dietaryregulation of thermogenesis.

Uncoupling protein 2 (UCP2) is a member of the uncoupling protein family. It isexpressed in the inner mitochondrial membrane and plays a role in the control offree radical production, oxidative damage, insulin secretion, and fatty-acidperoxide exportation. Although UCP2 expression occurs in several tissues, some ofits most remarkable functions are exerted in organs of difficult experimentalaccess, such as the central nervous system, particularly the hypothalamus and thepancreatic islets. In addition, due to its low levels of expression in themitochondrial membrane, studying UCP2 expression and function depends onspecific- and well-established methods. This chapter describes methods fordirectly assessing UCP2 expression and function in different tissues. Purifiedmitochondria preparations are used for enhancing the capacity of detection ofUCP2 protein or for evaluating the role of UCP2 in mitochondria respiration.Exposure of experimental animals to cold environment leads to increased UCP2expression, while reduction of its expression can be achieved directly bytargeting its mRNA with antisense oligonucleotides, or indirectly by targetingPGC-1alpha expression with antisense oligonucleotides.

PMID: 19426880

---------

[22] Curr Aging Sci. 2010 Jul;3(2):102-12.

Uncoupling protein-2 and the potential link between metabolism and longevity.

The discovery of novel uncoupling proteins (UCP2 and UCP3) over 10 years agoheralded a new era of research in mitochondrial uncoupling in a diverse range oftissues. Despite the research vigor, debate stills surrounds the exact functionof these uncoupling proteins. For example, the level of uncoupling, the mechanismand mode of action are all under-appreciated at this point in time. Our recentwork has used genetic mouse models to focus on the physiological relevance ofUCP2. We have used these mouse models to better appreciate the role UCP2 in humanhealth and disease. In this review we focus on new research showing that UCP2promotes longevity by shifting a given cell towards fatty acid fuel utilization.This metabolic hypothesis underlying UCP2-dependent longevity suggests that UCP2is critically positioned to maintain fatty acid oxidation and restrict subsequentoxidative damage allowing sustained mitochondrial oxidative capacity andmitochondrial biogenesis. These mechanisms converge within the cell to boost cellfunction and metabolism and the net result promotes healthy aging and increasedlifespan. Finally, UCP2 is a useful dietary and therapeutic target to promotelifespan and is an important mitochondrial protein connecting longevity tometabolism.

The long-term effects of uncoupled mitochondrial respiration by uncouplingprotein-2 (UCP2) in mammalian physiology remain controversial. Here we show thatincreased mitochondrial uncoupling activity of different tissues predicts longerlifespan of rats compared with mice. UCP2 reduces reactive oxygen species (ROS)production and oxidative stress throughout the aging process in different tissuesin mice. The absence of UCP2 shortens lifespan in wild-type mice, and the levelof UCP2 positively correlates with the postnatal survival of superoxidedismutase-2 mutant animals. Thus UCP2 has a beneficial influence on cell andtissue function leading to increased lifespan.

In the present investigation we describe the life span characteristics andphenotypic traits of ad libitum-fed mice that overexpress UCP2/3 (Positive-TG),their non-overexpressing littermates (Negative-TG), mice that do not expressionUCP2 (UCP2KO) or UCP3 (UCP3KO), and wild-type C57BL/6J mice (WT-Control). We alsoincluded a group of C57BL/6J mice calorie-restricted to 70% of ad libitum-fedmice in order to test partially the hypothesis that UCPs contribute to the lifeextension properties of CR. Mean survival was slightly, but significantly,greater in Positive-TG, than that observed in Negative-TG or WT-Control; meanlife span did not significantly differ from that of the UCP3KO mice. Maximal lifespan did not differ among the ad libitum-fed groups. Genotype did notsignificantly affect body weight, food intake, or the type of pathology at timeof death. Calorie restriction increased significantly mean and maximal life span,and the expression of UCP2 and UCP3. The lack of difference in maximal life spansamong the Positive-TG, Negative-TG, and UCP3KO suggests that UCP3 does notsignificantly affect longevity in mice.

PMID: 18854208 [PubMed - indexed for MEDLINE]

------

[25] Science. 2006 Nov 3;314(5800):825-8.

Transgenic mice with a reduced core body temperature have an increased life span.

Reduction of core body temperature has been proposed to contribute to theincreased life span and the antiaging effects conferred by calorie restriction(CR). Validation of this hypothesis has been difficult in homeotherms, primarilydue to a lack of experimental models. We report that transgenic mice engineeredto overexpress the uncoupling protein 2 in hypocretin neurons (Hcrt-UCP2) haveelevated hypothalamic temperature. The effects of local temperature elevation onthe central thermostat resulted in a 0.3 degrees to 0.5 degrees C reduction ofthe core body temperature. Fed ad libitum, Hcrt-UCP2 transgenic mice had the samecaloric intake as their wild-type littermates but had increased energy efficiencyand a greater median life span (12% increase in males; 20% increase in females).Thus, modest, sustained reduction of core body temperature prolonged life spanindependent of altered diet or CR.

Share this post

Link to post

Share on other sites

Dean -- thank you for putting together all that research and thoughtful analysis - great information.

Hear, hear! I, too, want to once again express my gratitude for the large amount of in-depth digging as well as valuable lighter dips into CR- and health-related science on the Forums. Your return to the CR community has been even more productive and valuable than it was during your first tenure, and we should all be grateful for your contributions.

I have even contacted [Arlan Richardson,] the researcher doing this ongoing study of DR in mice which may be of interest to people here. He was nice enough to respond, but didn't seem to think CE was a factor. Professor Arlan says:

Hollozy’s study showed that cold exposure did not change lifespan and in a study done at San Antonio (Ikeno et al.) they showed housing mice 1 vs 4 had the same lifespan whether fed AL or DR (in fact the DR mice group housed lived a little, but not significant, longer)

I have not tracked down those references.

Huh ...

"Holloszy's study" refers to the classic "rats with cold feet" experiment, with which Dean opened this thread. But "Ikeno et al" is quite another matter — and quite a surprise!

A quick search reveals (1):

This study examined the effect of housing density on the longevity-extending and disease-delaying actions of calorie restriction (CR). Singly or multiply housed (four per cage) mice were either fed ad libitum (AL) or were on CR beginning at 2 months. All CR mice were fed 40% less food than were multiply housed AL mice [not, NB, 40% less food than matching CRS mice -MR. To wit:]mice were randomly distributed into four groups: ALM, fed AL and housed four mice per cage; ALS, fed AL and housed one mouse per cage; CRM, fed 60% of the food intake of ALM mice and housed four mice per cage; and CRS, fed 60% of the food intake of ALM mice and housed one mouse per cage. To measure the amount of food consumption, the amount of chow removed from the cage hopper and the spillage (the chow on the bottom of the cage) were weighed weekly ... ... Thus, when compared to their housing density-appropriate control group, CRS mice received only 42% of the food intake of ALS mice, whereas CRM mice received 60% of the food intake of ALM mice [as, in absolute terms, did CRS mice -MR]. ...Averaged across the life span, ALS mice consumed approximately 40% more food than did ALM mice. ... Month-to-month variation in food intake was considerable, but was similar in both housing groups. This variation was probably due to variation in temperature of the colony room, because food intake was inversely related to room temperature (data not shown). ... CRS mice weighed about 20% less than CRM mice ... despite the fact that CRS and CRM mice consumed the same amount of food .. indicat[ing] that CRS mice were unable to store in body mass as many of the calories they consumed as were the CRM mice. Thus, proportionately more of the calories consumed by the CRS mice were expended in metabolism and maintenance of body temperature than were in CRM mice.

CR increased median longevity by 19%, and housing density had no effect on this increase. [Not quite true: see below]. CR also reduced neoplastic lesions in both housing groups, but ... A striking observation was that singly housed CR mice showed a greater reduction in the incidence of presumptively fatal neoplasms and in the severity of neoplasms than did CR mice housed multiply. There are several possible explanations for this difference. First, previous studies have shown a graded and proportionate reduction in induced cancers and the degree of CR (refs). CRS mice were more restricted in relation to their singly housed AL counterparts than were CRM mice in relation to their AL counterparts. [MR: I don't really buy this, but there it is]. Furthermore, the fact that CRS mice weighed 20% less than CRM mice indicates that CRS mice were unable to store in body mass as many of the calories they consumed as were the CRM mice. Thus, proportionately more of the calories consumed by the CRS mice were expended in metabolism and maintenance of body temperature than were in CRM mice ... [so] it is possible that cell division was reduced more in the CRS group or that the partitioning of energy utilization between growth and heat generation favored heat generation more in the CRS group than in the CRM group. ...

Despite their greater reduction in cancer incidence, CRS mice did not live longer than CRM mice. [Again, this rather understates the actual data:]

[Can't get the survivorship Table to reproduce, but the numbers are, for median and maximum (tenth-decile) survivorship (95% CIs in brackets):

ALS: 925 (839, 73) --> 1034 (1002, 1099)

ALM 935 (861, 983) --> 1062 (992, 1135)

CRS 1080 (957, 1135) --> 1252 (1209, 1362)

CRM 1133 (931, 1284) --> 1368 (1326, 1452)

[surprisingly, then, the warmer CRM mice, who weighed more and were slightly less protected from cancer than the CRS mice actually lived slightly longer! (yes, yes, the confidence intervals overlap — but just barely, and there were only 30 mice per subgroup). This is even more surprising, as group housing in CR mice can be dangerous, due to fighting over food. if you look at the survival curves, they were in lockstep until ≈900 d, similar to both cohorts' median LSs: this is consistent with the lesser cancer protection and apparently slower aging of the warmer CRS mice -MR]

Singly housed AL mice ate 40% more food than did multiply housed AL mice, but weighed the same and lived as long as multiply housed AL mice. These results indicate that CR can extend life span as effectively in multiply as in singly housed mice, even though housing density can differentially affect the cancer-reducing effect of CR.

The finding that the multiply housed CR mice that gained weight more slowly lived the longest is consistent with them ingesting fewer calories than their shorter lived cohabitants who gained weight more rapidly. In contrast, the finding that singly housed mice that gained weight the most rapidly lived longest would suggest that factors that enabled them to store a limited and fixed amount of energy more efficiently enabled them to outlive their counterparts who were less efficient at energy storage. Nevertheless, the fact that neither the median longevity nor its variance differed between the singly and the multiply housed CR groups indicates that, whatever the reason for the different relationships between weight gain and longevity in the two groups, this relationship did not impact the overall longevity-extending effect of CR.

Wow, that came up with a remarkable, relevant, and highly surprising study, Gordo! How fortunate you asked. That's a real puzzler under any "Keep Cool, CRONie!" hypothesis, BAT or otherwise. However, the broad fact of Calories trumping temperature on maximum LS despite a better anti-cancer benefit in cold mice is broadly consistent with the Koizumi & Walford study, where the warmer mice got more cancer (again, largely lymphoma), had a shortermedian LS, but similar maximum: absolute Calories are the key to the anti-aging effect.

Share this post

Link to post

Share on other sites

Dean, thanks for the additional info about skeletal muscle non-shivering thermogenesis (smNST), very interesting. There still may be an important link between BAT and smNST, I alluded to this earlier, but the quotes from the study (PMID 26993316):

Change (Δ) in BAT DEE as a result of cold stress was estimated to be 4 ± 3 kcal/day; this accounted for merely 1 % of the total change (Δ) in whole-body EE (352 ± 372 kcal/day,

In other words, when it comes to direct thermogenesis, BAT is practically doing NOTHING AT ALL (which still seems a little hard to believe in light of all the prior research and PET scans we've seen).

Share this post

Link to post

Share on other sites

Michael, that study (PMID: 16424282) IS indeed a good read. I'm sure Dean will dig in and do a much better job of commenting on it than I could. But just at a glance, a few thoughts popped into my head. For one thing, it seems like a glaring omission that they did not report the housing temps for this study when that was of critical importance. Both CR groups were 40% CRed, but the singly housed mice weighed 20% less because they were burning up extra calories to stay warm, which in one sense could mean they were the equivalent of "60% CRed" or maybe the multiply housed mice were also cold exposed (we don't know) making them the equivalent of 60% CRed and the singly housed mice %80 CRed. This study is further evidence that many if not most CR rodent studies have been poorly controlled (i.e. did not properly consider thermo-neutrality).

Next they found that CR w/CE resulted in substantially lower cancer incidence -- that sounds pretty good for CE, however the cold mice died more frequently of "mouse pneumonia". If translated to humans, this might actually be a pretty good trade off. That said, I think it's fair to say that no human (other than possibly Dean) is going to do CE 24/7 like these mice, so I'm really not sure how relevant these observations are (I doubt anyone has done "intermittent CE" studies on mice, but that would certainly be interesting). I would also love to see how the CRS vs. CRM comparison would go if the CRS mice were given just enough extra food so that their body weights matched the CRM cohort (effectively keeping calorie balance constant and only having one variable, temperature, changing).

Additionally, Dean previously referenced other studies showing CR w/CE in certain mouse genotypes had substantial life extending benefits beyond CR alone, taking that into consideration, and the fact that in this study lifespans were about the same with or without CE, its still looking pretty good for CE from my perspective. Furthermore, from A. Richardson's ongoing study description "two-thirds of the 41 recombinant inbred (RI) lines of mice studied either did not respond or showed reduced lifespan when fed 40% DR", then you have the primate CR studies showing little or no life extension beyond that of obesity avoidance... makes me want to look at going beyond CR alone.

But at the end of the day, I think CR/CE/special diets/Exercise are all only capable of fairly limited life extension. We need much bigger technological advances than anything discussed here if we are ever going to cheat death. Speaking of which, happy Easter everyone! ;)

Edited March 29, 2016 by Gordo

Share this post

Link to post

Share on other sites

Before getting to this latest study, I made a modest but I think interesting update to my recent big post on sarcolipin, and in particular the section on mitochondrial uncoupling. I added discussion of this study [26]. Below is the updated section. The underlined part is the new stuff, and the rest is mostly rewording:

Tantalizingly, quite a number of studies suggest mitochondrial uncoupling might be associated with increased longevity. Studies [22][23] found that increased UCP-2 expression results in increased longevity in rodents, and this may be associated with body temperature regulation [25], although this result is controversial [24]. Similarly suggestive, [26] found that mice chronically treated with a mild mitochondrial uncoupling agent that works independently of the UCPs (a chemical called 2,4-dinitrophenol or DPN) resulted in mice who ate more, weighed less, had lower measures of fasting glucose, triglycerides, insulin and oxidative damage to DNA, and most importantly, lived longer than controls - mean and maximum (oldest 10%) lifespan were extended by 7% and ~11%, respectively. And in this post, I talk about the evidence that increased expression of UCP proteins is associated with greater human longevity.

Regardless of the mechanism of mitochondrial uncoupling, and regardless of whether uncoupling is occurring in BAT, skeletal muscle, other organs, or some combination, it appears to be happening as a result of cold exposure, and have the potential to be health and lifespan promoting. The importance of mitochondrial uncoupling for thermogenesis in humans in response to cold exposure is demonstrated by [19], in which resting metabolic rate was increased by 76kcal/day as a result of mild cold exposure (80 hours @ 16 °F), and about half of that increase appeared to result from mitochondrial uncoupling.

While I think [26] is really interesting, I should note that it was done in a not especially long-lived strain of mice, so that is one caveat that should be considered about the results.

Share this post

Link to post

Share on other sites

Tantalizingly, quite a number of studies suggest mitochondrial uncoupling might be associated with increased longevity. Studies [22][23] found that increased UCP-2 expression results in increased longevity in rodents

First, [22][23] are not "studies," but a study [23] and a review covering [22] and related research. Second, [23] did not find that increased UCP-2 increased LS in normal, healthy animals: it found that "The absence of UCP2 shortens lifespan in wild-type mice, and the level of UCP2 positively correlates with the postnatal survival of superoxide dismutase-2 mutant animals." As Michael Rose once said,

"Until you show me that you can postpone aging, I don't know that you've done anything," sniffs Michael R. Rose, geneticist at the University of California. "A lot of people can kill things off sooner, by screwing around with various mechanisms, but to me that's like killing mice with hammers -- it doesn't show that hammers are related to aging."

Nor is reducing the severity of the severely-shortened postnatal survival of genetically screwed-up mice anything for normal, healthy people to get excited about.

The authors don't mention their actually relevant results in the abstract, which are that "No difference between survival curves of hUCP2 Tg mice [ie, mice given an extra dose of UCP-2],(n = 14) and WT controls was observed (n = 19, χ2 0.00085, df 1, P > 0.05; Fig. 4B), and mean survival age of hUCP2 mice was not different compared with WT controls [n = 19, 20.93 ± 2.65 vs. 23.95 ± 1.19 mo, not significant (ns)]." In fact, that may be being a little too strict: it may merit noticing that there was a numeric increase in median LS in mice given an extra dose of UCP-2, but without any effect on maximum, and while thre does seem to be some difference in the survival curve, it clearly shows that (a) the curve of the controls was not very rectangular, suggesting there was too much mortality in the controls, likely due to the metabolic morbidity of standard obesogenic lab feeding practices: "Mice and rats were kept under standard laboratory conditions with free access to standard chow food and water" — ie, true, literal ad libitum feeding (and no access to exercise); and (b) even granted that, the advantage came entirely in the first ≈20-22 mo, showing it was entirely related to premature mortality. The null hypothesis is pretty obviously obesity-avoidance — or the authors' own, more stickler's reading of "no difference."

although this result is controversial [24].

(I've intentionally rearranged this in my reply: your original order of wording made it sound as if (24) contrasted with (25), upon which it really doesn't touch: rather, it's related to (23)). It's not actually controversial, since the authors of (23) themselves acknowledge that there's "nothing to see here" in their hUCP2 Tg mice, which is the relevant finding. In any case, (24) is a properly-done LS study on UCP-2-overexpressing mice, with a nice rectangular control survival curve (and their food intake was properly measured, unlike in ((23)), and as I discussed here, it found no effect at all: zip, zero, zilch.

that this is just I and this may be associated with body temperature regulation [25]

I, too, am intrigued by (25), but again, this intervention increased mean but not maximum LS.

Similarly suggestive, [26] found that mice chronically treated with a mild mitochondrial uncoupling agent that works independently of the UCPs (a chemical called 2,4-dinitrophenol or DPN) resulted in mice who ate more, weighed less, had lower measures of fasting glucose, triglycerides, insulin and oxidative damage to DNA, and most importantly, lived longer than controls - mean and maximum (oldest 10%) lifespan were extended by 7% and ~11%, respectively.

Share this post

Link to post

Share on other sites

As I said, the longevity promoting effects of UCP proteins are pretty tentative and controversial, so you are right to be skeptical of positive benefits. But it seems relatively well-supported that extra energy expenditure (and therefore calories metabolized) as a result of higher mitochondrial uncoupling doesn't shorten lifespan, as your "calories, calories, calories" mantra would suggest it should. Right?

I'll talk more about this in my upcoming post about the newly uncovered Ikeno et al paper, so feel free to hold your response until that post. And of course you are more than welcome in the meantime to go back and comment on either my cold exposure albatross post or the bulk of my sarcolipin post, rather than just the small part of it that focused on mitochondrial uncoupling, which I do appreciate your cogent comments about.

--Dean

Share this post

Link to post

Share on other sites

I've got two big posts for this thread in the pipeline (on Ikeno et al and on Speakman's body of work). But I had to put those on hold to report on this new study [1], hot off the presses (popular press account). In fact, it's very related to several ongoing threads on these forums, so I think I'll be making several other posts as well to point to this one. It relates to four topics near and dear to our hearts, and germane to this thread - exercise, immune system function, cancer and cold exposure.

Unlike a lot of the studies we discuss, with many moving parts, this one was relatively straightforward, at least in its results. Researchers divided mice into individual cages, some with running wheels and some without. Unfortunately, they don't report the housing temperature, except to say (in the supplemental material) the mice were housed in a "thermo-stated environment", which I interpret to mean a temperature-controlled room. Since they didn't mention temperature explicitly, it seems almost certain the housing temperature was around 'normal laboratory conditions', i.e. 21-22 °C, which as we know is pretty chilly for mice - more on that below. The mice were allowed to run voluntarily (or not for the control mice) for four weeks. Then the mice were injected with various cancer promoting agents to induce cancer. After the injection, a group of the initially-sedentary mice were given a running wheel, to see how starting exercise after cancer initiation would impact the outcome. All the mice were then monitored to see how their tumor burden and blood chemistry changed over time.

What they found was quite dramatic. Giving mice free access to a running wheel reduced tumor burden by about 60% in both sexes, across range of ages (young, adult, older), and across a variety of different types of cancers, including lung cancer, liver cancer and skin cancer. But only if exercise was performed prior to cancer initiation - picking up the exercise habit after cancer had started didn't help slow tumor growth. And this effect wasn't a result of reduced food intake or weight loss in the exercisers (i.e. crypto-CR). The both sedentary and running mice we given ad lib access to food, and the running mice ate enough to compensate for the calories spent exercising, and so didn't lose weight. In fact, they ended up avoiding the weight loss exhibited by the cancer-ridden sedentary mice, and so weighed slightly more than the sedentary controls by the end of the study.

In short, the mice with access to the running wheel ate more, burned it off with lots of exercise (see below for just how much) so weighed virtually the same, and enjoyed dramatically slower cancer growth and tumor burden than sedentary control mice. Just to be graphic and to show how dramatic the effect was, check out these representative pictures of lung tumors taken from mice in the sedentary (CON - left) and exercise (EX - right) groups (note: the ugly black stuff is the tumor tissue):

So just how much were the mice running? It turns out the answer is a heckof a lot. The male mice in this study ran four miles (6.8 km) per day on the voluntary running wheel. That is virtually an identical distance to the male mice of the same strain in this study [2] (which found exercise increases the growth of new neurons - another great benefit!), which in addition to distance also reported how many minutes per day the mice ran. It turns out that to log 4 miles per day, the mice spend about 550 minutes per day chugging away on the running wheel. That's over 9 hours the mice spent exercising, and is coincidentally almost exactly how much I exercise per day, mostly spent gently pedalling away at my bikedesk, which now that I think about it seems quite analogous to a mouse on a running wheel. It's neat when things work out like that☺.

So what was the mechanism by which cancer was so improved in these chilly little exercise fanatics? That's where things get even more interesting. Through a series of experiments, the researchers determined it was the result of an exercise-induced increase in both epinephrine and Interleukin-6 (IL-6), which in turn boosted the immune systems (specifically, natural killer or NK immune cells) of the running mice so as to attack the cancer.

This is a cool discover for several reasons. First, the obvious one - it's good to slow the growth of cancer! But also, one concern we've been discussing a lot around here lately (e.g. here and here) is the potential negative impact of CR on immunity, and in particular our ability to fight off infections / invaders after they've gotten a foothold in our bodies. That is exactly the problem this study addresses and appears to provide an answer for. Namely, lots of low-intensity exercise coupled with mild cold exposure appears to dramatically improve the immune system's ability to tag and fight off foreign invaders - in this case cancer cells.

OK you might be saying - maybe I'll buy that nearly continuous, low-intensity exercise is great for boosting important immune system cell types, preventing/slowing cancer and (as a bonus) promoting neurogenesis (e.g. [2]). But besides your inference that the mice in this study were housed at normal lab temperatures and therefore were thermally stressed on top of the exercise, what's the link to cold exposure?

First off - as we've seen several times in this thread, in studies like Koizumi & Walford (PMID 9032756), and Ikeno et al. (PMID 16424282 - my post about which is in the works, stay tuned...) , mice housed in cool conditions exhibit dramatically less cancer mortality than mice housed at thermoneutrality - i.e. the temperature they are comfortable at. So it's very likely the tumor burden would have been even worse in the sedentary and in the exercising mice if they'd been house in warm conditions rather than the "normal" lab temperature presumably employed in this study.

But what's most relevant and exciting about these results vis a vis cold exposure are the details of the mechanism - i.e. elevated epinephrine and IL-6. Anyone who has been paying attention will know that cold exposure elevates epinephrine. In fact, epinephrine is the primary signalling molecule the body uses to orchestrate its response to cold exposure. I strongly suspect an increase in circulating epinephrine is the likely cause of the increase in resting heart rate (43 → 52 BMP) I've noticed since starting serious cold exposure.

As for interleukin-6, it's funny, in the post where I discuss CE's impact on the immune system, I noticed that PMID: 25338085 found humans who exercised at 0°C exhibited a reduction in a bunch of inflammatory markers, including "Interleukin-2, IL-5, IL-7, IL-10, IL-17, IFN-γ, Rantes, Eotaxin, IP-10, MIP-1β, MCP-1, VEGF, PDGF, and G-CSF", relative to people who exercised at normal room temperature (22 °C). At the time I thought it strange that one of the few interleukins I'd heard of (IL-6) wasn't on the list. It turns out IL-6 is often one of the 'good guys', particularly for muscles, unlike many of the other interleukins, which are pro-inflammatory and implicated in a number of diseases of aging, particularly when chronically elevated.

In fact, IL-6 isn't just left unchanged by cold exposure - it is increased in both rats [4] and humans. Regarding humans, from that same post, PMID 8925815 found IL-6 was increased (along with bunch of immune system cell types - see the post for the list) in humans as a result of chronic (6 weeks) of cold exposure, and PMID 10444630 found cold exposure after exercise boosts IL-6 levels in humans as well. This study I just uncovered [3], found the same thing - cold exposure after exercise increased (+30%) the already elevated level of IL-6 that resulted from exercise, as opposed to IL-6 dropping precipitously (-69%) when subjects were exposed to warm conditions after exercise. Here is the dramatic graph from [3] showing the post-exercise IL-6 levels in cold and warm subjects during the 90-minutes after exercise:

What's really interesting is the new light these results shed on the mechanism by which cold exposure reduces cancer. Previously I thought (and perhaps Michael did as well) that CE reduced cancer mortality simply by reducing body temperature like CR does, which in turned simply reduced cellular proliferation rate and therefore cancer progression. Sort of like how plants grow more slowly in cold weather - metabolic processes, including cancer growth, just slow down when it's cold. But these results suggest that reduced cancer mortality as a result of CE and/or exercise is likely a result of active upregulation of immune system function, in particularly increased natural killer (NK) cells, triggered by elevated epinephrine and IL-6. In short, this appears to be yet another pathway by which CE can improve health & longevity, both independently and synergistically with exercise. And I can't help but note for Michael's benefit that this beneficial CE pathway is quite independent of "obesity-avoidance".

So what have we learned from all this? I'm tempted to summarize it as "Dean was right"☺. But let me be a little more explicit. The evidence suggests that exercise coupled with cold exposure, or cold exposure alone, can reduce cancer growth and boost important aspects of immune system function (e.g. natural killer or NK cells) by increasing circulating levels of norepinephrine and interleukin-6.

This finding is particularly interesting and relevant given concerns about CR's potential deleterious effects on immunocompetence, and the new human CR study Michael announced a short time ago to investigate the effects of CR on the immune system. I hope I'm not disqualified from participating in the new study as a result of my dietary intake, parts of which Michael characterizes as "stupid high". I'd really like to see how my immune system compares with other CRers.

Share this post

Link to post

Share on other sites

Here is a new one [1] from Al (thanks Al!) that goes in the other direction (i.e. warm rather than cold exposure) with deleterious effects on important markers of health and longevity.

It tested 10 men in two experiments, where each served as their own control by repeating each of the conditions. The men started out fasting before each session began. The conditions were either resting for 40 minutes or a vigorous workout for 40 minutes, at either normal room temperature (22 °C) or warm temperature (31 °C) - making for a total of four different conditions. After each condition men either ate an ad lib meal consisting of unlimited ham and cheese sandwiches (average consumed meal equalled 1000kcal), or a standard oral dose of glucose 75g (300kcal) as part of an oral glucose tolerance test (OGTT). They measured glucose and insulin both during exercise, and after the meal or glucose challenge.

They found that either resting or exercising at warm temperatures resulted in higher serum glucose and higher serum insulin relative to exercising at room temperature. Here are the two most relevant graphs for glucose (left) and insulin (right). Sorry for the interfering overlay...:

As you can see, the black bars (warm conditions) are in each case worse (higher) than the corresponding normal temperature conditions. The glucose graph is particularly interesting. The area under the curve for glucose was slightly (non-significantly) lower after exercising than after resting at normal room temperatures. But cumulative glucose level was higher during and after exercising in warm temperatures relative to normal temperature exercise - and in this case the effect was significant. Notice I said during and after exercise. I was particularly surprised to see that glucose was a lot worse during exercise in warm temperatures, as can be seen dramatically in this graph, showing the time course of glucose:

As you can see, exercising in normal temperature conditions resulted in no elevation in glucose during exercise in a fasted state. In contrast, exercising at 31 °C resulted in a significant elevation in glucose during exercise relative to both resting and exercising in normal temperature.

In short, warm conditions appeared to impair glucose metabolism. This dovetails nicely with all the evidence presented previously in this thread pointing out how cold exposure improves glucose metabolism and insulin sensitivity, discussed in detail here. A prime example is [2], which found:

In other words, in people with BAT (likely due to chronic cold exposure), short-term cold exposure improves glucose metabolism and insulin sensitivity. It's unclear whether BAT is causing these improvements in humans directly, or is simply a marker for other cold adaptations (e.g. elevated sarcolipin in muscle cells to burn calories as heat) that are doing the real heavy lifting.

But whatever the mechanism, once again we see that warm is bad and cold is good when it comes to important markers of health and longevity.

Share this post

Link to post

Share on other sites

A post by Al on serotonin, thermoregulation and insomnia [1] promoted me to look into the connection. Al's study seemed to suggest that cold exposure may deplete serotonin, and as a result impair sleep, at least in mice. That link suprised me, since I'm finding I'm sleeping better these days since starting cold exposure, despite reducing the temperature in my room while sleeping.

It turns out that while cold exposure does indeed result in a drop in serotonin in rodents, the same (thankfully) doesn't appear to be true in humans, at least not to any large extent. Study [2] found only a small, non-significant drop in circulating serotonin as a result of cold exposure in sixty healthy men:

Nevertheless, the several times I've continued hard-core cold exposure (with my Cool Fat Burner vest) right up until bedtime, I've found my heart rate elevated (likely due to elevated epinephrine) and I had difficulty falling asleep. Difficulty falling asleep is very unusual for me - I usually have trouble staying asleep, a problem that seems to have disappeared since I started serious cold exposure.

So to make sure I can fall asleep, my practice of late has been to back off my degree of cold exposure for an hour or two prior to bedtime. This allows me to drop off to sleep easily. Then, since my bedroom is somewhat chilly, I can continue cold exposure while sleeping soundly, even getting back to sleep easily after my midnight trip to the bathroom.

--Dean

----------

[1] Sleep. 2015 Dec 1;38(12):1985-93. doi: 10.5665/sleep.5256.

Insomnia Caused by Serotonin Depletion is Due to Hypothermia.

Murray NM, Buchanan GF, Richerson GB.

Abstract

Study Objective:

Serotonin (5-hydroxytryptamine, 5-HT) neurons are now thought to promotewakefulness. Early experiments using the tryptophan hydroxylase inhibitorpara-chlorophenylalanine (PCPA) had led to the opposite conclusion, that5-HT causes sleep, but those studies were subsequently contradicted byelectrophysiological and behavioral data. Here we tested the hypothesis thatthe difference in conclusions was due to failure of early PCPA experimentsto control for the recently recognized role of 5-HT in thermoregulation.

PCPA treatment reduced brain 5-HT to less than 12% of that of controls.PCPA-treated mice housed at 20?C spent significantly more time awake thancontrols. However, core body temperature decreased from 36.5?C to 35.1?C.When housed at 33?C, body temperature remained normal, and total sleepduration, sleep architecture, and time in each vigilance state were the sameas controls. When challenged with 4?C, PCPA-treated mice experienced aprecipitous drop in body temperature, whereas control mice maintained anormal body temperature.

Conclusions:

These results indicate that early experiments using para-chlorophenylalaninethat led to the conclusion that 5-hydroxytryptamine (5-HT) causes sleep werelikely confounded by hypothermia. Temperature controls should be consideredin experiments using 5-HT depletion.

PMID: 26194567

---------

[2] Neuropsychobiology. 1993;28(1-2):37-42.

Lowering of body core temperature by exposure to a cold environment and by a5-HT1A agonist: effects on physiological and psychological variables and bloodserotonin levels.

The present study was designed to compare the effects of a pharmacologicallyinduced decrease in body core temperature to the effects observed with loweringof body temperature by exposure to a cold environment. Our special interest wasthe involvement of 5-HT in thermoregulatory responses. Sixty healthy malevolunteers were randomly assigned to one of the following conditions: exposure tonormal ambient temperature (28 degrees C) and placebo, exposure to cold ambienttemperature (5 degrees C) and placebo, or normal ambient temperature and 10 mg ofthe partial 5-HT1A agonist ipsapirone. As indicators of physiological responsesto lowering of body temperature, tympanic temperature, skin temperature, EMA,metabolic rate, and heart rate were monitored and saliva cortisol levels andperipheral 5-HT concentrations were determined. In addition, ratings on ambienttemperature, thermal discomfort, and feelings of irritability were obtained.While lowering of body core temperature was associated with markedcounterregulations (decrease of skin temperature, increase in EMA and metabolicrate) and feelings of discomfort, this was not observed with ipsapirone. Anincrease in cortisol levels was primarily observed in the ipsapirone group andwas not reflected by respective changes in whole blood or platelet 5-HTindicating that brain and platelet 5-HT are not related.

Share this post

Link to post

Share on other sites

Here is my long promised post in reply to Michael's post about Ikeno et al [1], which he seems to interpret as suggesting (to quote Michael):

That's a real puzzler under any "Keep Cool, CRONie!" hypothesis, BAT or otherwise...

[A]bsolute Calories are the key to the anti-aging effect.

First off, I think I understand why a cursory reading of [1] might lead someone to that interpretation. But I aim to show through more careful analysis in this post that Michael is mistaken. In fact, I aim to demonstrate that Ikeno et al [1] shows just the opposite - it provides strong support for the "Cool CR is good for health/longevity" hypothesis, and provides strong evidence against the absolute calories mantra Michael is so fond of.

To accomplish this challenging reversal of Michael's interpretation, I will do a head-to-head comparison between Ikeno et al [1] with one of the important papers which kicked this whole thread off - Koizumi & Walford [2], discussed previously here and here, which as you'll recall found that the benefits of CR were completely obliterated if the CR mice were housed at a thermoneutral temperature (30 °C) relative to CR at normal lab temperature (21 °C).

I will show the data between the two studies [1] and [2] lines up very consistently, and together the data and insights provided by the two studies support the "cool CR" hypothesis remarkably well. This will be a bit of a complicated story, but I'll walk you through it and I promise the payoff (new insights) at the end will be worth the effort to follow along.

First, a few assumptions:

The housing temperature of [1] is assumed to have been around "normal" lab temperature (21 °C). Why the heck didn't they report housing temperature? This is an egregious oversight on the parts of the authors. Not only wasn't temperature reported, but it looks like it varied too - since the authors say food intake varied seasonally, likely as a result of temperature variations in the animal housing facility. What the heck - wasn't their colony room temperature controlled!?

Month-to-month variation in food intake was considerable, but was similar in both housing groups. This variation

was probably due to variation in temperature of the colony room, because food intake was inversely related to room

temperature (data not shown).

The housing 4 mice per cage in [1] (and thereby permitting the mice to stay warmer by huddling) was approximately thermally equivalent to elevating their housing temperature to about the same as the 30 °C used in [2]. The large impact of group housing on temperature regulation of mice is backed up by [3], which found BAT activity was 50% lower in dual-housed mice relative to mice housed alone. The effect would be even bigger if housed 4-to-a-cage like in [1]. The near equivalence of 4-per-cage housing and 30 °C housing in terms of thermoregulation will be supported directly below in the head-to-head comparison by comparing food intake and body weight, so it's more than just an assumption.

Both [1] and [2] used the same mouse strain (C57BL/6J). Therefore their lifespans should be reasonably comparable across studies if the mice were treated the same.

Study [1] used male mice and [2] used female mice. From a large longevity study done [5] on this strain of mice (1000 males & females) to compare male vs. female longevity, it was found that male C57BL/6J live about 100 days longer than female mice on average. So I'm going to spot each group of mice in the Walford [2] study 100 extra days of lifespan to make it a fair lifespan comparison. You'll see once again this us supported by the data.

But before diving into the head-to-head comparison, a couple uncontroversial but important observations from [1] relevant to these discussions:

The cool (singly-housed) mice in [1] ate 40% more than "warm" (multiply housed) mice, but weighed the same, as a result of being chilly in single cages rather than being able to huddle. Despite eating 40% more, the cold mice lived just as long as the warm mice. Another example where eating extra calories doesn't reduce health or longevity, as long as it's burned off generating heat. Of course, since they were both (literally) AL-fed, these two groups' longevity wasn't terrific.

The cool CR mice in [1] ate the same amount as "warm" CR mice, weighed 20% less and lived the same mean and max lifespan as the warm CR mice. Here once again we see that cold exposure, even when combined with extreme, near-starvation CR - almost 60% restriction (i.e. only 40% as much food) relative to comparable controls (Singly-house AL mice), were able to live as long (both mean and max) lifespan as the warm CR mice. So even when you pound mice with cold-exposure on top of very severe CR, mice still aren't at a lifespan disadvantage relative to warm CR mice. But as I'll show below, you can do even better than lifespan parity with warm CR by cool housing mice and not cutting their calories to the point where the mice are nearly starving.

Not only were the cool CR mice in [1] nearly starved of sufficient calories @ 60% CR, they were also likely lacking in other nutrients as well. It was actually a food restriction rather than a calorie restriction experiment. In true and careful CR experiments (e.g. Koizumi & Walford [2] to which I'll compare), the mice in the CR group are fed extra vitamins, minerals and protein to keep their health and lifespan from getting cut short due to nutrient deficits. Study [1] did not take this precaution, and it is quite apparent the cool-house CR mice suffered the consequences, as I'll discuss more below.

The cool-housed CR mice were nearly immune to cancer. Only 4% of the cool-housed CR mice died of cancer, while 36% of the warm-housed CR mice died of cancer.

The cool-house CR mice in [1] died of infection - 50% died of pneumonia, compared with 10% of the warm-house CR mice, likely due to the poor cool-housed CR mice having a very compromised immune system due to near starvation-level net calorie restriction coupled with frank nutrient deficits. Personally, relative to a high(er) risk of cancers, many types of which have no effective treatment, I'd much prefer a higher risk of a condition I can avoid (pneumonia) via more calories, better nutrition and/or a simple vaccine. So even if my argument below is wrong, and there is an unavoidable tradeoff between lower cancer but higher infections as a result of chronic cold exposure, I still think it's rational to pick cold exposure (i.e. chose higher pneumonia risk over higher cancer risk).

Those are the major takeaway messages - cold exposure didn't hurt, either when the mice were allowed to get fat (feeding AL) or when kept thin. The cold CR mice were virtually immune to the #1 killer of lab rodents - cancer. But they were probably close to starvation and malnourished, and so their immune system was depressed making them unusually susceptible to pneumonia, which is avoidable and treatable in humans.

You may at this point be perfectly reasonable in questioning the above interpretations and conclusions. It's good to be skeptical. But in the following I aim to convince you.

I will now do a head-to-head comparison between this Ikeno et al study [1] Koizumi & Walford [2], using the following two graphs created from the data in the two papers. A couple comments about the notation in the graphs:

The groups referenced by 'I' (e.g. "Cool AL-I") refer to a group of mice from the Ikeno [1] study.

When I say 'cool' in reference to a group of mice, it means "housed at normal room temperature (~21 °C) with one mouse to a cage" - this was definitively stated to be the situation in Koizumi & Walford, and is presumed to have been the temperature in Ikeno et al. The food intake and body weight data will indeed strongly suggest this was the case for both groups.

When I say 'warm' in reference to a group of mice, it means "housed at 30 °C" in the case of Koizumi & Walford groups, and "housed 4-to-a-cage at normal room temperature" in the case of Ikeno et al groups.

Just a reminder - the Koizumi & Walford lifespans have been bumped up by 100 days to account for sex differences in longevity.

OK on to the data. This first graph compares the food intake, body weight and "longest lived 10%" lifespan of the cool-housed control animals in both the Ikeno (I) and Koizumi & Walford (W) studies.

As you can see, the control animals in the two studies had nearly identical weights (about 40g) and identical sex-adjusted lifespans (1050 days). The Ikeno controls were truly fed AL (35g/wk) while the Walford controls were reportedly fed 20% less that what they would otherwise eat if given free access to food (33g/wk), in order to prevent obesity. 20% less than 33g is 27g/wk. So interestingly, we see that the cool-housed Ikeno control mice ate a lot more, weighed the same, and lived the same as the Walford controls. This suggests if anything the mystery temperature in the Ikeno study was less that the room temperature used in the Walford study. This will come up again in the discussion of the CR conditions below. It also undermines Michael's contention that "it's abolute calories" that counts - since the Ikeno controls atemore, weighed the same and lived just as long as the Walford controls.

The above comparison of the two control groups is mostly to show the relative parity / comparability between the two studies once we adjust for the 100 day lifespan advantage of male mice of this strain. Same weight → same lifespan in the control groups after adjusting for known sex differences in longevity.

Now on to the interesting CR data comparison between the two studies, using the following identically-structured graph:

Now we have 4 sets of columns, representing the food intake, body weight and longevity of four sets of mice - warm and cool-housed mice from both the Ikeno and the Walford studies.

First let's look at the comparison of the "warm" groups from the two studies, in the left two sets of bars. The Ikeno warm-CR group ate a little more, weighed a little more, and lived nearly identical lifespan to the Walford warm-CR group. This data again points against Michael's "calories, calories, calories" hypothesis, since eating more, and even weighing more, didn't lead to a reduced lifespan in the Ikeno warm-CR mice, as that hypothesis would predict.

But things get really interesting when we compare the cool-CR groups from the two studies, in the right two sets of bars, and especially compare them with the warm-CR groups. First look at the cool-CR Ikeno group (Cool CR-I). They ate at least as much as the two warm-CR groups, but weighed significantly less than both of the others and lived just as long as the other two groups. So they weren't at a longevity disadvantage as discussed above.

But further, my contention (as described above), is that this Cool CR-I group was on the verge of starvation / malnutrition as a result of too little food and a deficiency of vitamins/minerals/protein for their cold-boosted metabolic requirements. By my account, because they were kept in the cold, they should have lived longer than the two warm CR groups, but unfortunately their lifespan was cut short by pneumonia due to a malnutrition-induced depressed immune system I talked about above.

Support for this interpretation of the Cool CR-I data comes from the rightmost set of bars - the cool-housed mice in the Walford study (Cool CR-W). Like the Cool CR-I mice, the Cool CR-w mice were kept cold. But unlike the Cool CR-I mice, the Cool CR-W mice were allowed more food (which was also enriched with vitamins, minerals and protein) than the warm CR mice to make up for the extra calories they were burning as a result of cold exposure. As a result, the body weight of the Cool CR-W mice remained close to the weight of the two warm-housed CR groups (~21g), rather than drop into the near-starvation "danger zone" that the Cool CR-I mice fell to (~18-19g). Further support that the Cool CR-I mice were near starvation level is the fact that even the 65% CR mice in the classic W&W paper [4] that adorns the CRS website weighed about 20g, which is more than the poor Cool CR-I mice in [1].

As a result of avoiding malnourishment and immune system suppression, the Cool CR-W mice had a much longer sex-adjusted lifespan - 1450 days compared with ~1250 for the other three CR groups. Like the Cool CR-I mice, very few of the Cool CR-W mice died of cancer - cool housing kicks butt against cancer. Unfortunately the Walford study [2] doesn't break down what the non-cancer causes of death were in their mice. But Walford et al did test immune system status directly and found:

At no age did energy restriction or housing temperature change the percentages of [the counts and ratios in serum, bone marrow and spleen of a bunch of different immune cell types - DP] CD4 + or CD8 + cells, CD4 + /CD8 + ratios, the percentage of Fas positive cells, the percentage of Fas positive CD4 + or the percentage of Fas positive CD8 + cells in bone marrow and spleen.

So in other words, the well-nourished thin (but not emaciated) and cold-exposed mice in Walford's cool CR group (Cool CR-W) had fully functional immune systems, and so avoided the infections than killed the nearly-starving Cool CR-I mice early, and so could take full advantage of the cancer-preventing benefits of cold-exposure, and so lived a lot longer than the warm CR mice in either study, and a lot longer than the cool but starved CR mice in the Ikeno study.

So in short, what this shows is that:

CR in warm temperatures sucks for lifespan relative to cool-CR, because cool-CR does a tremendous job at preventing cancer.

But cool-CR only manifests it's health/longevity advantage if you allow the cool-CR group to eat enough extra calories (and nutrients) to maintain their weight at a "slim" rather than "concentration camp" level, and to allow them to avoid a compromised immune system.

Put another way Cool-CR is good, but it requires eat sufficient extra calories to support both thermogenesis and the anabolic biochemical necessary to synthesize new cells in the immune system, generate new BAT tissue, new bone tissue and increase the number and size of mitochondria in muscle cells (PMID 25713078). A new exciting study about a novel, non-insulin/IGF-1-mediated anabolic pathway that may be involved in this increased maintenance and synthesis resulting from CE will be the topic of my next post.

So in closing, rather than undermining the "cold-exposure + enough calories to remain slim = optimal health & longevity" hypothesis as Michael suggests, a careful reading of Ikeno et al [1], in combination with Walford et al [2], provides strong support for the hypothesis.

A group of 1,052 C57BL/6J mice (296 males and 756 females) was kept underwell-defined, clean laboratory conditions from the age of 6 weeks until naturaldeath. The survival curves of males and females (computer-produced 3, 4, and 5parameter curves, Gompertz curve histogram) were established and shown to followa logistic function. The average life-span amounted to 878 plus or minus 10 daysfor males and 794 plus or minus 6 days for females. These values distinctlyexceed comparable values given in the literature. They are attributed tofavorable conditions of animal care and to supposed alterations in geneticbackground. A genetic drift in sex-dependent mean survival time occurred in thegenetically unstable C57BL/6J strain between 1966 and 1970. Before this drift,the males died sooner; after it, they lived longer.